CN102105487A

CN102105487A - CRKL targeting peptides

Info

Publication number: CN102105487A
Application number: CN2009801292540A
Authority: CN
Inventors: W·阿拉普; P·J·门茨; R·帕斯夸里尼
Original assignee: University of Texas System
Current assignee: University of Texas System
Priority date: 2008-06-20
Filing date: 2009-06-19
Publication date: 2011-06-22
Also published as: CR20110034A; JP2011525491A; EP2303913A2; WO2009155556A3; PE20110309A1; MX2010014173A; CL2010001498A1; WO2009155556A2; EP2303913A4; ECSP10010729A; IL210053A0; CA2728763A1; US20110189095A1; BRPI0915718A2; CO6331291A2

Abstract

Provided are methods and compositions for selectively targeting CRKL through the use of targeting peptides. Selective targeting of secreted CRKL through the use of a targeting peptide may be used, for example, in the treatment of cancer to deliver a chemotherapeutic compound, fusion protein, or fusion construct to a cancer cell or tissue.

Description

The peptide of target CRKL

The application requires the right of priority of the U. S. application submitted on June 20th, 2008 number 61/074,423, and its whole disclosures do not have institute and abandon ground and incorporate this paper especially by reference in full into.

The present invention is making under the fund PC050442 in National Institute of Health and Department of Defense under the support of United States Government.Government enjoys rights more of the present invention.

Background of invention

1. invention field

The present invention relates to the field of the targeted of molecular medicine and treatment and detection reagent.More specifically, the present invention relates to of the evaluation of selectivity target on cancer with the new peptide sequence of treatment and detection cancer.

2. the description of association area

The therapeutic treatment of a lot of human disease states is subjected to the toxic restriction of general of used therapeutical agent.Cancer therapeutic agent especially demonstrates low-down therapeutic index, and when reagent concentration is high a lot of unlike the concentration that is used for the kill tumor cell, and mushroom healthy tissues for example skin and marrow can be affected usually.Be used for composition and method by exploitation to the cancer cells targeted, more specifically, by using and being present in the cancer cells surface but not being present in target bonded antibody or tumor-homing peptide (tumor-homingpeptide) in the healthy tissues, can greatly promote treatment for cancer and diagnosis exactly.This type of target must have minimum homology with other cell surface molecule, its be difficult to found.

Recently, use phage demonstration library to develop selective system in a kind of body, to identify the organ or tissue's target peptide in the mouse model system.This type of library can produce like this: random oligonucleotide is inserted among the proteic cDNA of coding phage surface, produce to reach 10 ⁹Individual arrangement shows set (Pasqualini and Ruoslahti, 1996, people such as Arap, 1998 of the phage particle of unique peptide; People such as Pasqualini, 2001).Application of bacteriophage shows after the library in the mouse vein that carries tumour, reclaims phage from tumor xenogeneic graft, and characterize can selectivity to the peptide of the target tumor of tumor-homing.The phage (Pasqualini and Ruoslahti, 1996) that the sequence of the selectively targeted peptide of expressing based on the phage outside surface, recovery can be optionally gone back to the nest to the vescular bed of different mouse organ or tissue.In those tumor-homing peptides each all is bonded to the acceptor in tumor cell surface selective expression or rise.

Therapeutical agent and the organ or tissue that wants that is transported to being connected of target peptide in the mouse model system with causing this reagent selectivity.Chemotherapeutics and pro-apoptotic peptide are to being arranged in the toxic decline of mouse model general (people such as Arap, 1998a, the 1998b that targeted that tumor vessel forms the acceptor of property vascular system causes the remarkable rising of therapeutic efficiency and carries tumour; People such as Ellerby, 1999).

CRKL (No. 10 conditioning agents of the proteic chicken tumor disease poison of kinases sample) is a kind of adaptin (adapter protein), and it is the homologue of oncogene v-crk.It comprises 1 SH2 structural domain and 2 placed in-line SH3 structural domains.Intracellular CRKL participates in map kinase and 6 integrin-mediated approach (people such as Li, 2003; People such as Uemura, 1999).In addition, CRKL has carcinogenic potentiality.

At present, use general chemotherapy and radiation method conventional treatment cancer patients.But this type of therapy often is subjected to the puzzlement of well-known side effect and limited effect.The new composition and the method that obviously need be used for targeted treatment and diagnostic reagent.

Summary of the invention

The present invention is used for the optionally method and composition of target excretory CRKL by using the target peptide to provide, thereby has overcome defective of the prior art.By using target peptide selectivity target excretory CRKL to can be used for for example treatment for cancer, so that carry chemotherapy compound, fusion rotein or fusion constructs to cancer cells or tissue.

In order to obtain the understanding about cross-cell membrane signal conduction mechanism aspect in the cancer, the functional protein that the inventor begins to disclose in the tumour heteroplastic transplantation model interacts.Inventor's inference: combined method (people such as Hajitou, 2006; People such as Arap, 2002; People such as Arap, 1998; People such as Arap, 2004; Pasqualini and Ruoslahti, 1996), for example may give a clue from phage demonstration random peptide library Continuous Selection in vivo, this is undertaken by imitating the agonic ligand-receptor combination under the tumor microenvironment situation.As shown in following examples, observe signal conductive protein CRKL and modulability (rather than part associativity) β in the cell ₁Specificity between the integrin extracellular domain interacts.Surprisingly, the inventor finds CRKL target β ₁(plexin-semaphorin-integrin, PSI) structural domain excite map kinase and promote cell growth and survival the extracellular clump shape albumen-brain signal albumen-integrin that is positioned at of integrin.Do not wish to be bound by any theory, these results support following viewpoint: in the process that activates the map kinase approach, for conditioning agent in the cell (protein that for example contains SH3), have as yet the not 6 integrin-mediated function from outside to inside of understanding.

One aspect of the present invention relates to the peptide of the isolating target tumor that comprises the CRKL binding motif, and described motif is defined as: length is 6-20 amino acid, with β ₁The similarity degree of the corresponding optimum match sequence alignment of integrin (SEQ IDNO:47) is at least 25%; And the length of wherein said target peptide is 100 amino acid or amino acid still less, and is bonded to the cell of expressing CRKL under physiological condition.Described CRKL binding motif and β ₁The similarity degree of the best-fit sequence alignment of integrin (SEQ ID NO:47) can be at least 40%, at least 50% or at least 60%.In some embodiments, described peptide has and is different from and β ₁The sequence of the best-fit sequence alignment of integrin (SEQ ID NO:47).In some embodiments, described CRKL binding motif can have and β ₁The best-fit sequence alignment in integrin (SEQ IDNO:47) PSI structural domain zone.Described CRKL binding motif can have and β ₁The best-fit sequence alignment in integrin (SEQ ID NO:47) PSI structural domain zone, described PSI structural domain zone is selected from amino acid 6 to 10; 10 to 29; 15 to 34; 18 to 37; 36 to 55; 39 to 58; 45 to 64; 94 to 113; 196 to 215; 198 to 213; 203 to 222; 244 to 263; 330 to 349; 377 to 396; 379 to 398; 380 to 399; 398 to 417; 400 to 419; 413 to 432; 447 to 466; 460 to 479; 460 to 479; 464 to 483; 469 to 488; 474 to 493; 475 to 494; 512 to 533; 519 to 538; 551 to 570; 574 to 593; 577 to 596; 579 to 598; 590 to 609; 596 to 615; 613 to 632; 615 to 634; 616 to 635; 644 to 663; 648 to 667; 663 to 682; 674 to 693; 682 to 701; 721 to 740; 727 to 746; With 779 to 798.In some embodiments, the CRKL binding motif has the sequence that is selected from SEQ ID NO:1 to SEQ ID NO:46.

In some embodiments, isolating peptide can be further defined as: can be with the cyclic peptide of annular form preparation, the peptide that for example has cysteine residues (" C ") at two ends, when needs, can provide described peptide with annular form by for example forming two-halfcystine (being Gelucystine).This type of cyclic peptide can have the meaning of particularly important: the disulfide linkage in the peptide makes it have remarkable stability for chemistry, heat or enzymatic degradation.This type of cyclic peptide may have the meaning of particularly important in treatment and diagnostic use, wherein relate to the problem that utilizability is poor, be easy to take place transformation period weak point in proteolysis and the body.

Described peptide can be connected to molecule; For example, described molecule can be an albumen, and described peptide can put together or merge to described albumen to form protein conjugate, wherein said protein conjugate is not naturally occurring albumen.Peptide can be positioned at proteic end.Described molecule can be that short natural death of cerebral cells reagent, anti-angiogenic agent, cytokine, cytotoxic reagent, medicine, chemotherapeutics, hormone, somatomedin, microbiotic, antibody or its fragment or strand, survival factors, anti-cell are transferred die agent, hormone antagonist, antigen, peptide, albumen, diagnostic reagent, radio isotope or preparation.Described molecule can be to be selected from following short natural death of cerebral cells agent: linear gramicidins, magainin (magainin), mellitin, alexin, cecropin (cecropin), (KLAKLAK) ₂(SEQ ID NO:48), (KLAKKLA) ₂(SEQ ID NO:49), (KAAKKAA) ₂(SEQ ID NO:50), (KLGKKLG) ₃(SEQ ID NO:51), Bcl-2, Bad, Bak, Bax and Bik.In some embodiments, described short natural death of cerebral cells agent is (KLAKLAK) ₂(SEQ ID NO:48).SEQ ID NO:48 can be made up of D amino acid.

In other embodiments, described molecule can be to be selected from following angiogenesis inhibitor reagent: thrombospondin, angiostatin, the pigment epidermal derived factor, angiotensin, the ln peptide, the fibronectin peptide, inhibitors of plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, Interferon, rabbit, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, the 2-methoxyestradiol, the proliferin associated protein, carboxylic amine triazole, CM101, Marimastat, the many vitriol of piperylene, angiogenin 2, Antibiotic TAN 420F, PNU145156E, 16K prolactin antagonist fragment, linomide (Linomide), thalidomide, pentoxifylline, genistein, TNP-470, Endostatin, taxol, Docetaxel, polyamine, proteasome inhibitor, kinase inhibitor, signaling peptides, accutin, cidofovir, vincristine(VCR), bleomycin, AGM-1470, platelet factor 4, MINOCYCLINE HCL, Endostatin XVIII, Endostatin XV, the terminal Hemopexin structural domain of the C-of matrix metalloproteinase-2, kringle 5 structural domains that human plasmin is former, the fusion rotein of Endostatin and angiostatin, the fusion rotein of kringle 5 structural domains that Endostatin and human plasmin are former, the monokine that interferon-induces (Mig), the fusion rotein of Mig and IP10, solubility FLT-1 (fins sample Tyrosylprotein kinase 1 acceptor), or kinases inserts minor structure domain receptor (KDR).Described molecule can be to be selected from following cytokine: interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-(IF-γ), IF-α, IF-β, tumour necrosis factor, GM-CSF (rHuGM-CSF).

Described peptide can be connected to macromolecular complex, for example virus, phage, bacterium, liposome, microparticle, magnetic pearl, yeast cell or mammalian cell.In some embodiments, described peptide is connected to virus, for example slow virus, papovavirus, adenovirus, retrovirus, AAV, vaccinia virus or simplexvirus.Described peptide can be connected to solid carrier, for example microtitration ware or microchip.

Another aspect of the present invention relates to the method for preparing construct, and it comprises: obtain peptide of the present invention, and described peptide is connected to molecule to prepare described construct.

Another aspect of the present invention relates to peptide, molecule or proteic method of sending the cell of targeted expression CRKL, said method comprising the steps of: obtain or by method for preparing according to peptide of the present invention, and described peptide is applied to cell colony, wherein said colony comprises the cell of expressing CRKL, thereby molecule or albumen are delivered to described cell.The cell of expressing CRKL can be in the experimenter, and peptide or albumen fusion constructs can be formulated in the pharmaceutically acceptable composition, and described composition can be applied to the experimenter.Described experimenter can the human experimenter.In some embodiments, this method is further defined as detection method, and described method comprises that also detection has been transported to the peptide of cell, molecule or albumen.Described experimenter can suffer from disease or illness, and described method can be further defined as methods of treatment.Described experimenter can have cancer, for example prostate cancer, mammary cancer, sarcoma, gingival carcinoma, tongue cancer, lung cancer, skin carcinoma, liver cancer, kidney, cancer eye, the cancer of the brain, leukemia, mesothelioma, neuroblastoma, a cancer, neck cancer, carcinoma of the pancreas, kidney, osteocarcinoma, carcinoma of testis, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, the cancer of the brain, colorectal carcinoma and bladder cancer.

The embodiment of discussing in the context of method of the present invention and/or composition can be applied to any other method described herein or composition.Therefore, relate to a kind of method or composition embodiment and also can be applicable to other method and composition of the present invention.

In the specification sheets of this paper, " one (a) " or " one (an) " can refer to one or more.In the claim of this paper, when " comprising (comprising) " when using with word, word " (a) " or " one (an) " can refer to one or more.

Unless offer some clarification on is only to refer to surrogate or surrogate objectionable intermingling, otherwise the term that uses in the claim " or " be used in reference to " and/or ", though disclosure support only refer to surrogate and " and/or " definition." another " used herein can refer to another one or a plurality of at least.

In this application, term " approximately " is used to represent numerical value, comprises equipment, is used to measure the intrinsic difference of error of the method for this numerical value, or be present in the difference among the research experimenter.

Other target of the present invention, feature and advantage will become obvious by following detailed description.But, should be appreciated that,, only provide and describe in detail and specific embodiment with interpretive mode though indicate preferred implementation of the present invention, because from this detailed description, multiple variation in the spirit and scope of the invention and modification will be tangible for those skilled in the art.

Description of drawings

The following drawings is the part of this specification sheets, comprises that these accompanying drawings are in order to further specify some aspect of the present invention.Can be by understanding the present invention better with reference to accompanying drawing and in conjunction with the detailed description of embodiment provided herein.

Figure 1A-C: the target of peptide and internalization in the cancer cells.Figure 1A, phage display peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) is bonded to the cell surface of DU145 cell.The phage clone of display sequence RGD-4C people such as (, 1998) Arap is as positive control; Fd-tet (not having the son of insertion) is as negative control.The bar post is represented the mean value ± standard deviation of triplicate coated plate.Figure 1B, the immunolocalization of tumor-homing phage on the cell surface of the KS1767 cell of non-penetratingization.Fig. 1 C and D, synthetic peptide YRCTLNSPFFWEDMTHECHAGG (SEQ ID NO:67)- _D(KLAKLAK) ₂Make and in the DU145 cell, internalization takes place, survey by cell survival as using WST-1 reagent and anti--annexin-VFITC antibody

Fig. 2 A-C: tumor-homing peptide and β ₁The sequence alignment of integrin (SEQ ID NO:13).Fig. 2 A, tumor-homing peptide YRCTLNSPFFWEDMTHECHA (SEQ IDNO:1) and clump shape albumen-brain signal albumen-integrin (PSI) structural domain (sequence area 26-78 residue) (SEQ ID NO:12) coupling.Fig. 2 B, the sequence alignment of all 8 β integrin-subunits (SEQ IDNO:14-21) and YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) peptide sequence.Fig. 2 C, all tumor-homing peptide (table 1) and β ₁The sequence alignment of integrin (SEQ IDNO:13).

Fig. 3 A-D: peptide combines with acceptor.Fig. 3 A, reorganization His-label C RKL (rCRKL), rCRKL-SH3 (N) structural domain and rCRKL-SH3 (C) structural domain are bonded to tumor-homing peptide-YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1).Fig. 3 B, reorganization His-label rCRKL, rCRKL-SH3 (N) structural domain and rCRKL-SH3 (C) structural domain are bonded to the synthetic peptide NSTFLQEGMPTSA (SEQ ID NO:23) corresponding to the zone in the PSI structural domain.Fig. 3 C, the reorganization gst fusion rotein, linear gst-PSI (the residue 48-62 that have prepared whole PSI structural domain (residue 22-82.SEQ ID NO:71-CLKANAKSCGECIQAGPNCGWCTNSTFLQE GMPTSARCDDLEALKKKGC), SEQ ID NO:72-TNSTFLQEGMPTSAR), ring type gst-PSI (residue 26-74, SEQ ID NO:72-CTNSTFLQEGMPTSARC), to be used in conjunction with measuring.Linear and ring type PSI deutero-zone all is bonded to CRKL.Fig. 3 D, the tumor-homing peptide is active in tumor-homing peptide (YRCTLNSPFFWEDMTHECHA with combining of rCRKL-SH3 (C) structural domain; SEQ ID NO:1), PSI deutero-peptide (NSTFLQEGMPTSA; SEQ ID NO:23) or show that the tumor-homing phage of SEQ ID NO:1 suppresses.The representative of bar post is from the mean value ± standard deviation in triplicate hole.

Interaction between Fig. 4 A-D:CRKL and the binding peptide.Fig. 4 A, the binding characteristic of rCRKL-SH3 (C) and tumor-homing peptide.Pro-＞Ala among the SEQ ID NO:1 is expressed as SEQ ID NO:25.The mutation analysis of CRKL SH3 (C) structural domain.Fig. 4 B, (it shows YRCTLNSPFFWEDMTHECHA to the tumor-homing phage; SEQ ID NO:1) and PSI deutero-phage (it shows CNSTFLQEGMPTSA C; SEQ ID NO:23) is bonded to the CRKL of reorganization.The representative of bar post is from the mean value ± standard deviation in triplicate hole.Fig. 4 C, CRKL SH3 (C) structural domain (SEQ ID NO:24) and the diagram of deleting mutant as the contrast that His-label recombinant protein produces.Preparation has also been tested 4 kinds of mutant: Δ 1 (deletion residue 236-256), Δ 2 (deletion residue 257-277), Δ 3 (deletion residue 278-293) and Δ SH3 (C) (deletion residue 236-293).Fig. 4 D, calmodulin binding domain CaM are positioned between the residue 236-277 of SH3 (C) structural domain.

Protein-protein interaction between Fig. 5: CRKL and the beta 1 integrin.Reorganization gst-PSI albumen suppresses CRKL and β in the concentration dependent mode ₁The combination of integrin (until 800nm).Beta 2 integrin alpha v β 3 and α v β 5 usefulness compare.Shown standard deviation from the mean value in triplicate hole.

The cell surface location of Fig. 6 A-B:CRKL.Fig. 6 A, the flow cytometry of the CRKL on the DU145 cell.Use monoclonal anti-CRKL ( ^*), anti--β ₁Integrin ( ^*) and resist-AHSG antibody (c, contrast) carries out immune labeled.Fig. 6 B, the transmission electron microscope of CRKL (TEM) analysis has shown the single CRKL-gold grain (arrow) on the cell surface.The DU145 cell fixation that will be used to study but penetratingization not.Use anti--CRKL polyclonal antibody in the research.Indicated scale.

The secretion of Fig. 7 A-C:CRKL.Fig. 7 A, various types of cancer cells of cultivating in serum free medium (SFM) are secreted the not CRKL of phosphorylation form.Figure B and C, the CRKL of extracellular form and influence cell proliferation and migration in the CRKL antibody and in the substratum.The control antibodies of using is as follows: anti--the IL11 acceptor, anti--AHSG, anti--grb2, anti-α ₆The antibody of integrin and pre-immunity.The representative of bar post is from the mean value ± standard deviation in duplicate hole.

Fig. 8 A-D:siRNA strikes the effect of low CRKL.Show cell proliferation (Fig. 8 A), adhered to (Fig. 8 B) and migration (Fig. 8 C).Shown standard deviation from the mean value in triplicate hole.Fig. 8 D, in cell proliferating determining, recombinant C RKL saves CRKL siRNA and strikes low cell.Use CRKL siRNA transfection DU145 cell 48 hours, and added recombinant C RKL to seedbed, China and foreign countries, hole then.Use WST-1 reagent to measure cell proliferation.

Fig. 9 A-D: cancer target and mechanical model.Fig. 9 A, tumor-homing phage or contrast phage (do not have the son of insertion, mutant (YRCTLNSAFFWEDMTHECHA; Or miscellaneous phage (#1:YRFCTSPFHEWHLENTDMCA SEQ IDNO:25); SEQID NO:26, #2:YRECTDSPHEFHLWNTMCAF; SEQ ID NO:27)) the external combination on rCRKL.Fig. 9 B-D goes back to the nest in target phage or the body of contrast phage construct in carrying dissimilar mice with tumor.When comparing, the tumor-homing phage preferentially is positioned tumour.Shown representative data from two independent experiments.

Figure 10: the target that carries in the mouse of tumour suppresses.Use contrast gst or reorganization gst-CRKL incubation tumor-homing phage in advance, use to the nude mice of the human tumor that carries size match (derived from DU145) then.Observe the inhibition of recombinant C RKL for pretreated tumor-homing phage.Shown result from two independent experiments.

Figure 11: handle tumor xenogeneic graft with the short natural death of cerebral cells peptide of synthetic tumor-homing.The formation of nude mice of the size match of human prostate cancer heterograft (derived from DU145) is carried in use.Use the short natural death of cerebral cells peptide YRCTLNSPFFWEDMTHECHAGG (SEQ ID NO:67) of synthetic tumor-homing- _D(KLAKLAK) ₂Observe the tumor growth of remarkable minimizing in the mouse of carrying tumour of handling.Compare with undressed animal, the YRCTLNSPFFWEDMTHECHA of equimolar amount (SEQ IDNO:1) or _D(KLAKLAK) ₂Do not demonstrate the difference (Student t-check, p＜0.001) on the gross tumor volume.

Embodiment

The invention provides support about the importance of signal conductive protein CRKL in the cell and the evidence of function, the peptide of target CRKL also is provided.Cytolemma evolved between intracellular matter and extracellular environment, go into closely with control (Conner andSchmid, 2003 of compartmentation; Cho and Stahelin, 2005).In order to keep this running balance, transmembrane receptor family mediates signal conduction (people such as Martin, 2002 of the twocouese of crossing over cell surface by the room and time framework of the complexity of transductory cascade; People such as Manning, 2002).Therefore, the proteic location of participation signal transduction has crucial meaning (Cho and Stahelin, 2005 for the specificity that cell response is provided; Mochly-Rosen, 1995).For example, under the environment of typical machinery transduction, by part in conjunction with the trigger cell surface receptor for example the integrin occurred conformation change, thereby realize and for example mutual communication of mitogen activated albumen (MAP) kinase pathways of signal transduction cascade; Conventional integrin ligands comprise at its extracellular domain extracellular matrix (ECM) albumen and at the cytoskeletal protein of its cell intracellular domain (people such as Martin, 2002; People such as Manning, 2002; Hunter, 2000; Pawsonand Scott, 1997; Blume-Jensen and Hunter, 2001).

In order to obtain the understanding about transmembrane signal transduction mechanism aspect in the cancer, the functional protein that the inventor begins to disclose in the tumor xenogeneic graft model interacts.Inventor's inference: combined method (people such as Hajitou, 2006; People such as Arap, 2002; People such as Arap, 1998; People such as Arap, 2004; Pasqualini and Ruoslahti, 1996), for example may give a clue from phage demonstration random peptide library Continuous Selection in vivo, this is undertaken by imitating the agonic ligand-receptor combination under the tumor microenvironment situation.Following data show, at intracellular signal conductive protein CRKL and modulability (rather than part associativity) β ₁Exist specificity to interact between the integrin extracellular domain.Surprisingly, the inventor finds CRKL target β ₁(plexin-semaphorin-integrin, PSI) structural domain excite map kinase and promote cell growth and survival the extracellular clump shape albumen-brain signal albumen-integrin that is positioned at of integrin.These results suggest 6 integrin-mediated from outside to inside the function as yet do not recognized of intracellular conditioning agent (protein that for example contains SH3) in activating the map kinase approach.

Consistent with the cumulative performance data that presents in this work, extracellular CRKL might have not revealed as yet effect in tumor microenvironment: it brings into play this effect by trigger cell propagation and migration.Because the external source rCRKL phosphorylation that the intracellular portion of CRKL itself also is added, so autocrine or paracrine factor that people can the extracellular CRKL of inference (excretory and/or release) may can be used as in the tumour play a role.These results have set up the uncommon novel association between signal transduction molecule and the cell adhesion acceptor, and wherein they can cause signal conduction incident from extracellular environment in the relation of cell surface.

Without wishing to be bound to any theory, based on integrin activated " spring tool (switchblade) " structural models (people such as Takagi, 2002), the inventor has supposed a kind of substituting approach, and wherein extracellular CRKL can activate integrin (becoming activated extended conformation from the crooked conformation of non-activity) by the PSI structural domain that is bonded to the beta 1 integrin chain.In the multistep model (Fig. 6) that this paper presents, the intracellular unphosphorylated CRKL of following digital proof is by the secretion of non-classical active transport (may be sub by the ABC-transhipment), and/or be released to by necrocytosis that (step 1), wherein its SH3 structural domain specificity is in conjunction with the PSI structural domain (step 2) of the beta 1 integrin on the tumor cell surface in the tumor microenvironment.In conjunction with after, the conformation of beta 1 integrin becomes stretching, extension (active) from bending, thereby cause the phosphorylation (step 3 and 4) and/or the map kinase approach (step 5-7) of the target protein of 6 integrin-mediated approach middle and lower reaches, and finally influence the migration and the propagation (step 8) of tumour cell.Consistent with these discoveries, have recently several pieces about other also can be in the cell that cell surface is detected molecule, comprise multiple nucleoprotein (Sinclair and O ' Brien, 2002; People such as Hovanessian, 2000), transcription factor (people such as Monferran, 2004) and stress response companion (people such as Arap, 2004; People such as Shin, 2003; People such as Mintz, 2003).Except a functional ligand-receptor interaction that in following examples, shows, between the signal transduction molecule of excretory that acts on extracellular environment and/or release and cell adhesion acceptor, may there be other functional ligand-receptor interaction, and may has general biological significance.

The invention provides the peptide of isolating target tumor, it comprises the CRKL binding motif, and described motif is defined as, and for example length is 6-20 amino acid, with β ₁The similarity degree of the corresponding optimum match sequence alignment of integrin (SEQ IDNO:47) is for example at least 25%; And wherein said target peptide length can be 100 amino acid or amino acid still less, and is bonded to the cell of expressing CRKL under physiological condition.Described CRKL binding motif and β ₁The similarity degree of the best-fit sequence alignment of integrin (SEQ ID NO:47) can be at least 40%, at least 50% or at least 60%.In some embodiments, described peptide has and is different from and β ₁The sequence of the best-fit sequence alignment of integrin (SEQ ID NO:47).In some embodiments, described CRKL binding motif can have and β ₁The best-fit sequence alignment in integrin (SEQ IDNO:47) PSI structural domain zone.Described CRKL binding motif can have and β ₁The best-fit sequence alignment in integrin (SEQ ID NO:47) PSI structural domain zone, described PSI structural domain zone is selected from amino acid/11 0 to 29; 15 to 34; 18 to 37; 36 to 55; 39 to 58; 45 to 64; 94 to 113; 196 to 215; 198 to 213; 203 to 222; 244 to 263; 330 to 349; 377 to 396; 379 to 398; 380 to 399; 398 to 417; 400 to 419; 413 to 432; 447 to 466; 460 to 479; 460 to 479; 464 to 483; 469 to 488; 474 to 493; 475 to 494; 512 to 533; 519 to 538; 551 to 570; 574 to 593; 577 to 596; 579 to 598; 590 to 609; 596 to 615; 613 to 632; 615 to 634; 616 to 635; 644 to 663; 648 to 667; 663 to 682; 674 to 693; 682 to 701; 721 to 740; 727 to 746; With 779 to 798.In some embodiments, the CRKL binding motif has the sequence that is selected from SEQ ID NO:1 to SEQ ID NO:46.In some embodiments, isolating peptide of the present invention can be further defined as cyclic peptide.

The present invention can use multiple CRKL binding peptide.Non-limiting the enumerating (SEQ ID NO:1-46) of CRKL binding peptide below is provided.

The tumor-homing peptide that table 1. and beta 1 integrin have similarity

Definition

Term " targeting moiety " comprises the polytype affinity reagent of bonded of specific position in the location that can be used for the specific position of enhancing substance in animal or enhancing substance and the animal, and described specific position comprises organ, tissue, specific cell type, illing tissue or tumour.Targeting moiety can comprise peptide, peptide mimics, polypeptide, antibody, antibody molecule, nucleic acid, fit and fragment.In some embodiments, targeting moiety is with the location of enhancing substance to the cell of expressing CRKL (being that CRKL combines with cell surface or combines with on every side extracellular matrix) in the extracellular.The selective binding of targeting moiety of the present invention (for example target peptide and variant thereof and fragment) is: targeting moiety is in conjunction with target (for example CRKL) and do not combine with uncorrelated albumen is remarkable.Even targeting moiety also with those in fact with target other protein binding of homologous not, as long as the fragment or the structural domain of the peptide target of this proteinoid and antibody have homology, then this targeting moiety still is considered to selective binding.In this case, should be appreciated that though to a certain degree cross reactivity is arranged, targeting moiety remains optionally with combining of target.Usually, can determine the degree of cross reactivity and separate with land with target.

" peptide of target tumor " is the peptide that comprises the CRKL binding motif, and described motif is defined as: length is 6-20 amino acid, with β ₁The similarity degree of the corresponding optimum match sequence alignment of integrin (SEQ ID NO:47) is at least 25%; And the length of wherein said target peptide is 100 amino acid or amino acid still less, and it is characterized in that selective fixed organ, tissue or the cell type of being positioned under physiological condition, and this comprises with extracellular CRKL specificity and combining.Can for example pass through hereinafter disclosed method mensuration selectivity location, wherein Jia Ding target peptide sequence is bonded to the albumen that is shown in the phage outside surface.

" experimenter " generally is meant Mammals.In some preferred implementations, the experimenter is mouse or rabbit.In preferred embodiment, the experimenter is the people.

CRKL (No. 10 conditioning agents of the proteic chicken tumor disease poison of kinases sample)

Chronic lymphocytic leukemia (CML) is the blood system malignant disease, and uncontrolled granulocyte propagation wherein takes place.Its feature is normally: karyomit(e) 9 and 22 mutual transposition, this can be indexed into Ableson (abl) proto-oncogene 3 ' end of breakpoint cluster region (bcr).This can produce chimeric bcr-abl gene, its coding p210.sup.bcr-abl fusion rotein, and this albumen is tumorigenicity, and is essential (people such as Szczylik, 1991 for the growth of CML cell; People such as Skorski, 1994; People such as Tari, 1994; People such as McGahon, 1994; People such as Bedi, 1994).

Autophosphorylation can take place at 177 tyrosine amino acid places of first exon that is present in bcr in bcr-abl albumen.When phosphorylation, the proteic bcr structural domain of bcr-abl is bonded to the proteic SH2 structural domain of growth factor receptors bonded albumen 2 (Grb2) joint.By the SH3 structural domain, Grb2 is bonded to people SOS1, and (this causes the proteic activation of ras for Son of sevenless 1, hSos1) GDP/GTP exchange factor.Bcr-abl albumen can also phosphorylation be present in 177 tyrosine amino acid in the normal bcr albumen.Believe that it also will be compound with Grb2 when normal bcr albumen during at 177 amino acids generation tyrosine phosphorylations.When bcr-abl albumen was expressed, tyrosine phosphorylation also took place in p46 and p52Shc (people such as Puil, 1994) albumen.Shown that also these Shc albumen and Grb2 are stably compound.Therefore, as if Grb2 has important effect (people such as Puil, 1994 in the protein mediated tumour of bcr-abl takes place; People such as Pendergast, 1993).

Have been found that also another kind of joint PROTEIN C rk sample (CRKL) is bonded to bcr-abl.Different with Grb2, CRKL is bonded to bcr-abl by the abl structural domain.By its SH3 structural domain, CRKL can also be bonded to hSos1, and this also can cause the proteic activation of Ras (people such as tenHoeve, 1994a and 1994b).Therefore, by Grb2 and CRKL joint albumen, the activation of bcr-abl albumen and ras associates, and the activation of known ras can cause tumour to take place.When the proteic expression of ras was suppressed, the propagation of CML cell also was suppressed.Therefore, bcr-abl albumen promotes that one of main path of CML propagation is by activating ras albumen people such as (, 1994 and 1995) Skorski.

The inventor finds, at intracellular signal conductive protein CRKL and modulability (but not part associativity) β ₁Observing specificity between the integrin extracellular domain interacts.Surprisingly, the inventor finds, is known as the CRKL target β of cell internal connection usually ₁(plexin-semaphorin-integrin, PSI) structural domain excite map kinase and promote cell growth and survival the extracellular clump shape albumen-brain signal albumen-integrin that is positioned at of integrin.Do not wish to be bound by any theory, these results support following viewpoint: in the process that activates the map kinase approach, for conditioning agent in the cell (protein that for example contains SH3), have as yet the not 6 integrin-mediated function from outside to inside of understanding.

Sequence alignment is analyzed

Sequence similarity is defined as two sequence identity between the nucleotide sequence, but must not represent that these two sequences have the common ancestor.For example, 25% the similarity meaning is in two nucleotide sequences, has 25 to be identical on 100 nucleotide positions." best-fit sequence alignment " among the present invention is defined as such sequential analysis: find the segmentation (part) or the overall comparison of the optimum matching of two or more sequences by using those of ordinary skills' known sequences comparison method.Those skilled in the art will know, and can use multiple algorithm to carry out sequence relatively, and for example BLAST is than equity.

Can use technology disclosed herein to analyze tumor-homing phage display peptide and β ₁Sequence alignment between the integrin.The peptide adapting software (Peptide Match software) of writing among the Perl 5.8.1 that is based on RELIC50 that the inventor uses.This program is based on predetermined residue window size, with from N to the C albumen end mode of residue displacement one by one, calculates the peptide sequence of affine selection and the similarity between the target protein sequence.Calculate the peptide-protein similar score of each residue based on BLOSUM62 amino-acid substitution matrix (making amendment) to consider the expression of rare amino acid.Can be at least 4 identical residues between peptide and the albumen section with threshold value setting, to distinguish the remarkable similarity of mating with non-specific background.

Albumen and peptide

In some embodiments, the present invention relates to comprise the novel compositions of at least a albumen or peptide.Albumen used herein or peptide generally are meant but are not limited to, from next about 200 albumen more than the amino acid of gene translation, until full length sequence; The polypeptide that about 100 amino acid are above; And/or about 3 to about 100 amino acid whose peptides.For convenience's sake, term " albumen ", " polypeptide " and " peptide " use on mutual alternative ground in this article.

In some embodiments, the size of at least a albumen or peptide can include but not limited to 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more a plurality of amino-acid residue.For example, the target peptide may reside in the fusion rotein to produce albumen.

In some respects, the size of the peptide of the target tumor that defines among the present invention can include but not limited to 5,6,7,8,9,10,11,12,13,14,15,16, and 17,18,19,20 amino-acid residues.In others, the size of the peptide of target tumor can include but not limited to 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28, and 29,30 amino-acid residues maybe can be from any scope of deutero-wherein.In some embodiments, can use length to be less than or equal to 20 amino acid whose peptides, perhaps length is 6-10 amino acid whose peptide.

" amino-acid residue " used herein is meant any naturally occurring amino acid, any amino acid derivative known in the art or any amino acid analog thing.In some embodiments, the residue of albumen or peptide is a successive, interrupts the sequence of amino-acid residue without any non-amino acid.In other embodiments, sequence can comprise one or more non-amino acid moieties.In concrete embodiment, the sequence of the residue of albumen or peptide can be interrupted by one or more non-amino acid moieties.

Correspondingly, term " albumen or peptide " comprises at least a or at least a modification or the unconventional amino acid whose aminoacid sequence that contains 20 kinds of common amino acids finding in the naturally occurring protein, amino acid described modification or unconventional includes but not limited to, Aad, 2-aminoadipic acid; EtAsn, the N-ethyl asparagine; Baad, the 3-aminoadipic acid; Hyl, oxylysine; Bala, Beta-alanine, beta-amino-propionic acid; AHyl, not-oxylysine; Abu, the 2-aminobutyric acid; 3Hyp, the 3-oxyproline; 4Abu, 4-aminobutyric acid, nipecotic acid; 4Hyp, the 4-oxyproline; Acp, 6-aminocaprolc acid; Ide, different bad ammonia element; Ahe, the 2-aminoheptylic acid; AIle, not-Isoleucine; Aib, the 2-aminoisobutyric acid; MeGly, sarcosine, sarkosine; Baib, the 3-aminoisobutyric acid; MeIle, N-methyl Isoleucine; Apm, the 2-diaminopimelic acid; MeLys, the 6-N-methyllysine; Dbu, 2,4-diamino-butanoic; MeVal, the N-methylvaline; Des relies the ammonia element; Nva, norvaline; Dpm, 2,2 '-diaminopimelic acid; Nle, nor-leucine; Dpr, 2, the 3-diaminopropionic acid; Orn, ornithine; And EtGly, Ethylglycocoll.

Can prepare albumen or peptide by any technology well known by persons skilled in the art, comprise by standard molecular biological technique expressing protein, polypeptide or peptide, from natural origin protein isolate or peptide, or chemosynthesis albumen or peptide.Disclosed Nucleotide and albumen, polypeptide and peptide sequence in the past, can from the known computerized data bank of those of ordinary skills, find corresponding to several genes.A kind of such database is a National Center forBiotechnology Information ' s Genbank and GenPept database (network address is ncbi.nlm.nih.gov).Can use the coding region of known technology amplification of technology disclosed herein or those of ordinary skills and/or expression known.Perhaps, the multiple merchant of albumen, polypeptide and peptide to sell goods be known to those skilled in the art.

Fusion rotein

Other embodiment of protein conjugate relates to fusion rotein.These molecules generally have the whole or substantial part of the peptide of target tumor, N or C-terminal be connected to second polypeptide or proteic all or part of.For example, fusions can be used to from the homing sequence of other species to allow recombinant expression protein in heterologous host.Another kind of useful fusions comprises and adds the immunologic competence structural domain, and antibody epitope for example is so that for example promote the purifying of fusion rotein.Near the removal of external polypeptide after the adding cleavage site will promote purifying at the fusion joint or it.Other useful fusions comprises the linkage function structural domain, for example from avtive spot, glycosylation structural domain, the cell-targeting signal of enzyme or stride the film district.In preferred embodiment, fusion rotein of the present invention comprises the peptide of the target LRP that is connected to treatment albumen or peptide.The example that can be incorporated into albumen in the fusion rotein or peptide comprises the albumen that suppresses cell, cytocidal albumen, short natural death of cerebral cells reagent, anti-angiogenic agent, hormone, cytokine, somatomedin, peptide medicine, antibody, Fab fragment antibody, antigen, receptor protein, enzyme, lectin, MHC albumen, cell adhesion protein and conjugated protein.These examples are not to be intended to limit, and consider within the scope of the invention, in fact any albumen or peptide can be incorporated in the fusion rotein that comprises the target peptide.The method that produces fusion rotein is well known to those skilled in the art.This proteinoid can be produced like this, and for example: use the bifunctional cross-linker by the chemistry connection, from new synthetic complete fusion rotein, the dna sequence dna of the target peptide of maybe will encoding is connected to coding second peptide or proteic dna sequence dna, the fusion rotein of The expressed then.

Protein purification

In some embodiments, albumen or peptide can be isolating or purifying.The protein purification technology is well known to those skilled in the art.These technology comprise, on a level, are polypeptide and non-polypeptide component with cell, tissue or organ homogenization and roughing out.Can use chromatography and electrophoretic technique to be further purified target protein or polypeptide, to reach partially or completely purifying (or being purified to even matter).The analytical procedure that is particularly suitable for preparing pure peptide is that ion-exchange chromatography, gel are got rid of chromatogram, polyacrylamide gel electrophoresis, affinity chromatography, immune affinity chromatographic and isoelectrofocusing.Be disclosed in United States Patent (USP) 5,206,347 by the proteic example of affinity chromatography purified receptor, its full content is incorporated this paper by reference into.The special effective means of purified peptide is fast liquid chromatography (FPLC), or high performance liquid chromatography (HPLC).

The albumen of purifying or peptide mean can with the composition of other component separating, wherein with respect to its natural obtainable state, described albumen or peptide are purified to any degree.Therefore, the albumen of isolating or purifying or peptide also refer to be free on albumen or the peptide that it can naturally occurring environment.Generally speaking, " purifying " refers to albumen or the peptide combinations of experience separation to remove various other components, and described composition keeps the biological activity of its expression in fact.When using term " purifying in fact ", this term is meant such composition, wherein albumen or peptide constitute the main component of said composition, for example constitute in the said composition about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more albumen.

According to the disclosure, the various methods that are used for the degree of purification of quantitative albumen or peptide are well known by persons skilled in the art.These methods for example comprise, measure the specific activity of active fraction, or pass through the amount of the in one's duty polypeptide of SDS/PAGE analysis and evaluation level.The preferred method that is used for the purity of evaluation stage part is the specific activity that calculates this grade part, and its specific activity with initial extract is compared, and calculates its degree of purification thus, assesses by " multiple purifying number ".Certainly, the effective unit that is used to represent active amount will depend on the particular analysis technology that is used to carry out purifying of selection, and whether expressed proteins or peptide show detectable activity.

The various technology that are suitable for protein purification are well known to those skilled in the art.These comprise, for example, use material such as ammonium sulfate, PEG, antibody or precipitate by thermally denature, carry out then: centrifugal; Chromatographic step, for example ion-exchange, gel-filtration, anti-phase, hydroxyapatite and affinity chromatography; Isoelectrofocusing; Gel electrophoresis; And the combination of these and other technology.As this area, know, believe that the order of carrying out each purification step can change, perhaps can omit some step, and still produce and be used to prepare in fact the albumen of purifying or the proper method of peptide.

Not about " always providing albumen or peptide " general requirement with the state of purifying.Really, consider that the product than the purifying in fact of low degree will have application in some embodiments.Can use the purification step of less combination, or by utilizing multi-form identical general purification scheme to finish partial purification.For example, recognize that the cation exchange column chromatography that uses HPLC equipment to carry out generally will produce than constructed bigger " multiple " purifying that uses the low-pressure chromatography system.Demonstrating may be aspect total recovery of protein product or keeping having advantage aspect expressed proteins active than the method for the relative purifying of low degree.

Affinity chromatography is the chromatographic program that depends on material to be separated and its specificity avidity between can specificity bonded molecule.This is the interaction of receptor-ligand type.Column material be by will in conjunction with one of right member with the insoluble matrix covalent coupling synthetic.Then, described column material can be from solution the specific adsorption material.By condition changing is carried out wash-out for bonded condition (for example, changing pH, ionic strength, temperature etc.) can not take place.Matrix should be itself not with any obvious degree adsorbed molecules, and the material that has quite widely chemistry, physics and thermostability.Part should carry out coupling in the mode that does not influence its binding characteristic.Part also should provide combination relatively closely.Also should be not destroy the mode eluted material of sample or part.

The synthetic peptide

Because its less relatively size can be according to routine techniques synthetic target peptide of the present invention in solution or on solid carrier.Multiple automated synthesiser can obtain from the commercial channel, and can use according to known program.Referring to, Stewart and Young for example, 1984; People such as Tam, 1983; Merrifield, 1986; Barany and Merrifield, 1979, above document is incorporated this paper all by reference into.Can be by the easily synthetic short peptide sequence of these class methods, normally about 6 to an about 35-50 amino acid.Perhaps, can use recombinant DNA technology, the nucleotide sequence of the peptide of the present invention of wherein encoding is inserted in the expression vector, and conversion or transfection enter proper host cell, and cultivate under the condition that is suitable for expressing.

Treatment or diagnosis conjugate

Use the targeting moiety that these methods identify can coupling or be bonded to multiple material, comprise treatment or diagnostic reagent, be used for the conjugate selectivity is delivered to the organ of wanting, tissue or the cell type of mouse model system.Embodiments of the present invention relate to disease or illness, preferred treatment for cancer.The peptide of target tumor of the present invention can be connected to molecule; For example, described molecule can be an albumen, and described peptide can put together with this albumen or merge to form protein conjugate, and wherein said protein conjugate is not naturally occurring albumen.Described peptide can place described proteic end.Described molecule can be short apoptosis reagent, angiogenesis inhibitor reagent, cytokine, cytotoxic reagent, medicine, chemotherapeutics, hormone, somatomedin, microbiotic, antibody or its fragment or one-sided love, survival factors, anti-apoptotic agent, hormone antagonist, antigen, peptide, albumen, diagnostic reagent, radio isotope or imaging agents.

The peptide of target tumor can be connected to macromolecular complex, for example virus, phage, bacterium, liposome, microsphere, magnetic pearl, yeast cell or mammalian cell.In some embodiments, described peptide is connected to virus, for example slow virus, papovavirus, adenovirus, retrovirus, AAV, vaccinia virus or simplexvirus.Described peptide can be connected to solid carrier, for example microtitration ware or microchip.

A. the conditioning agent of apoptosis

Apoptosis, or apoptosis are normal fetal developments, keep the running balance in the adult tissue and suppress requisite process people such as (, 1972) Kerr of oncogenesis.Proved that the albumen of Bcl-2 family and ICE-sample proteolytic enzyme are apoptotic important conditioning agent and the effectors in other system.Bcl-2 albumen (being found under the situation relevant) with follicular lymphoma in control at various apoptosis stimulated cells apoptosis with strengthen and to have vital role in the cell survival (people such as Bakhshi, 1985; Cleary and Sklar, 1985; People such as Cleary, 1986; People such as Tsujimoto, 1985; Tsujimoto and Croce, 1986).Recognize that now Bcl-2 albumen conservative in the evolution is the member of associated protein family, it can be classified as death agonist or dead antagonist.

Find after the Bcl-2, proved that Bcl-2 plays the effect of the necrocytosis that suppresses multiple stimulation initiation.Equally, know clearly also now that have Bcl-2 necrocytosis conditioning agent protein family, they have common structure and sequence homology.Shown family member that these are different or had similar function (for example, Bcl to Bcl-2 _XL, Bcl _W, Bcl _S, Mcl-1, A1, Bfl-1), or offset the function of Bcl-2 and promote necrocytosis (for example, Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

The example of short apoptosis agent has: linear gramicidins, magainin (magainin), mellitin, alexin, cecropin (cecropin), (KLAKLAK) ₂(SEQ ID NO:48), (KLAKKLA) ₂(SEQ ID NO:49), (KAAKKAA) ₂(SEQ ID NO:50), (KLGKKLG) ₃(SEQ ID NO:51), Bcl-2, Bad, Bak, Bax and Bik.In some embodiments, described short natural death of cerebral cells agent is (KLAKLAK) ₂(SEQ IDNO:48).SEQ ID NO:48 can be made up of D amino acid.

B. angiogenesis inhibitor

The propagation of tumour cell depends on a large amount of neonate tumour blood vessels to a great extent, and it follows the progress of cancer.Therefore, the target that uses angiogenesis inhibitor reagent to suppress the formation of neovascularity and existing blood vessel destroy be suggested as effectively with the method for relative avirulent treatment tumour (people such as Arap, 1998; People such as Arap, 1998; People such as Ellerby, 1999).Multiple angiogenesis inhibitor reagent and/or vasoinhibitor are known (for example, Folkman, 1997; Eliceiri andCheresh, 2001).

In some embodiments, the present invention can adopt using of the targeting moiety that operationally is coupled to angiogenesis inhibitor reagent, described angiogenesis inhibitor reagent for example is, thrombospondin, angiostatin, the pigment epidermal derived factor, angiotensin, the ln peptide, the fibronectin peptide, inhibitors of plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, Interferon, rabbit, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, the 2-methoxyestradiol, the proliferin associated protein, carboxylic amine triazole, CM101, Marimastat, the many vitriol of piperylene, angiogenin 2, Antibiotic TAN 420F, PNU145156E, 16K prolactin antagonist fragment, linomide (Linomide), thalidomide, pentoxifylline, genistein, TNP-470, Endostatin, taxol, Docetaxel, polyamine, proteasome inhibitor, kinase inhibitor, signaling peptides, accutin, cidofovir, vincristine(VCR), bleomycin, AGM-1470, platelet factor 4, MINOCYCLINE HCL, Endostatin XVIII, Endostatin XV, the terminal Hemopexin structural domain of the C-of matrix metalloproteinase-2, kringle 5 structural domains that human plasmin is former, the fusion rotein of Endostatin and angiostatin, the fusion rotein of kringle 5 structural domains that Endostatin and human plasmin are former, the monokine that interferon-induces (Mig), the fusion rotein of Mig and IP10, solubility FLT-1 (fins sample Tyrosylprotein kinase 1 acceptor), or kinases inserts minor structure domain receptor (KDR).Described molecule can be to be selected from following cytokine: interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-(IF-γ), IF-α, IF-β, tumour necrosis factor or GM-CSF (rHuGM-CSF).

C. cytotoxic reagent

Chemotherapy (cytotoxicity) reagent can be coupled to target peptide of the present invention and be used for the treatment of multiple excess proliferative or tumour formation property morbid state, comprises cancer.Chemotherapy (cytotoxicity) reagent with potential use includes but not limited to, 5 FU 5 fluorouracil, bleomycin, busulfan, camptothecine, carboplatin, Chlorambucil, cis-platinum (CDDP), endoxan, gengshengmeisu, daunorubicin, Zorubicin, the estrogen receptor wedding agent, Etoposide (VP16), farnesyl protein transferase inhibitors, gemcitabine, ifosfamide, mustargen, melphalan, mitomycin, nvelbine, nitrosourea, Plicamycin, procarbazine, raloxifene, Tamoxifen, taxol, Temozolomide (the water form of DTIC), trans platinum, vinealeucoblastine(VLB) and methotrexate, vincristine(VCR), or above-mentioned these any analogue or the variant of deriving.Most chemotherapeutics drop in the following category: alkylating reagent, metabolic antagonist, antitumor antibiotics, corticosteroid hormone, mitotic inhibitor and nitrosoureas, hormone reagent, miscellaneous reagent, and any analogue or the variant of deriving.

Chemotherapeutics and medication, dosage etc. be well known to those skilled in the art (referring to, for example " Physicians Desk Reference ", Goodman﹠amp; Gilman ' s " ThePharmacological Basis of Therapeutics " and see " Remington ' sPharmaceutical Sciences ", the 15th edition, 1035-1038 and 1570-1580 page or leaf, incorporate relevant portion into this paper by reference), and can unite use with the present invention in light of the disclosure herein.Certainly, depend on the experimenter's who is treated situation, some variations can take place in dosage.Under any circumstance, the personnel of responsible administration will be identified for the suitable dose of individual subjects.Certainly, all dosage described herein and reagent all are illustrative rather than restrictive, and for specific patient or application, the technician can use other dosage or reagent.Expect in addition between these points all dosage or also can be used for the present invention by its deutero-scope.

D. alkylating reagent

Alkylating reagent is and the medicine of genomic dna direct interaction with prevention cell proliferation.This based chemotherapy medicine representative influences the reagent in all stages of cell cycle, that is, they are not phasic specificities.Alkylating reagent can include but not limited to, mustargen, ethyleneimine, methyl melamine, alkylsulfonate, nitrosourea or triazine.They include but not limited to: busulfan, Chlorambucil, cis-platinum, endoxan (cyclophosphamide, cytoxan), Dacarbazine, ifosfamide, mustargen (mechlorethamine, mustargen) and melphalan.

E. metabolic antagonist

Metabolic antagonist destroys the synthetic of DNA and RNA.Different with alkylating reagent, they influence the cell cycle S phase specifically.Metabolic antagonist can be divided into different classifications, for example folacin, pyrimidine analogue and purine analogue, and relevant inhibition compound.Metabolic antagonist includes but not limited to, 5 FU 5 fluorouracil (5-FU), cytosine arabinoside (Ara-C), fludarabine, gemcitabine and methotrexate.

F. natural product

Natural product generally is meant at first from natural resource and separates and be accredited as the compound with pharmacological activity.This compounds, its analogue and derivative can separate from natural resource, by chemosynthesis, or by any technology reorganization generation well known by persons skilled in the art.Natural product comprises for example following classification: mitotic inhibitor, antitumor antibiotics, enzyme and biological response modifier.

Mitotic inhibitor comprises that plant alkaloid and other can suppress cell fission or the required albumen synthetic natural agent of mitotic division.They play a role in the specified phase of cell cycle.Mitotic inhibitor comprises, for example, and Docetaxel, Etoposide (VP16), Vumon, taxol (paclitaxel), taxol (taxol), vinealeucoblastine(VLB), vincristine(VCR) and vinorelbine.

Toxoid (taxoid) is from ash tree yewtree (formal name used at school: separate and the next relevant compound of a class bark Taxus brevifolia).Toxoid includes but not limited to compound, for example Docetaxel and taxol.Taxol is in conjunction with tubulin (its binding site and vinca alkaloids used site different) and promote the assembling of microtubule.

Vinca alkaloids is a kind of plant alkaloid, and it is accredited as has pharmaceutical active.They comprise the compound such as vinealeucoblastine(VLB) (VLB) and vincristine(VCR).

G. microbiotic

Some microbiotic have antimicrobial and cellular cytoxicity activity simultaneously.These medicines are also by chemically inhibitory enzyme and mitotic division or change cytolemma disturb DNA.These reagent are not phasic specificities, so they all played a role in all stages of cell cycle.Cytotoxic antibiotic example includes but not limited to, bleomycin, gengshengmeisu, daunorubicin, Zorubicin (doxorubicin, Adriamycin), Plicamycin (mithramycin) and darubicin.

H. miscellaneous cytotoxic reagent

The miscellaneous cytotoxic reagent that does not fall into cocategory includes but not limited to, the urea of platinum complex mixture, amerantrone, replacement, methyl hydrazine derivative, amsacrine, altheine enzyme and vitamin A acid.The platinum complex mixture comprises the compound such as carboplatin and cis-platinum (cis DDP).Exemplary amerantrone is a mitoxantrone.The urea of exemplary replacement is a hydroxyurea.Exemplary methyl hydrazine derivative be procarbazine (the N-methyl hydrazine, MIH).These examples are not restrictive, consider within the scope of the invention, any known cytotoxicity, suppress cell or cytocidal reagent can be connected to the target peptide and be applied to organ, tissue or the cell type of target.

I. imaging agents and radio isotope

In some embodiments, targeting moiety of the present invention can be connected to the imaging agents that is used for imaging and diagnosis multiple ill organ, tissue or cell type.The method that a lot of suitable imaging agents and being used to are connected to albumen or peptide be known in the art (referring to, for example United States Patent (USP) 5,021,236 and 4,472,509, the two incorporates this paper by reference into).Some methods of attachment comprise uses the metal-chelating mixture, and for example organic sequestrant for example is connected to the DTPA (United States Patent (USP) 4,472,509) of albumen or peptide.Albumen or peptide can also exist coupling agent for example under the situation of glutaraldehyde or periodate with enzyme reaction.Exist under the situation of these coupling agents or by preparing conjugate with the fluorescein mark with the isothiocyanic acid reactant salt.

The limiting examples that potential can be used as the paramagnetic ion of preparation comprises chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (IIII), and wherein gadolinium is particularly preferred.Other situation for example in the x-ray imaging useful ion include but not limited to lanthanum (III), gold (III), plumbous (II), particularly bismuth (III).

The potential radio isotope that can serve as picture or therapeutical agent comprises astatine ²¹¹, ¹⁴Carbon, ⁵¹Chromium, ³⁶Chlorine, ⁵⁷Cobalt, ⁵⁸Cobalt, copper ⁶⁷, ¹⁵²Eu, gallium ⁶⁷, ³Hydrogen, iodine ¹²³, iodine ¹²⁵, iodine ¹³¹, indium ¹¹¹, ⁵⁹Iron, ³²Phosphorus, rhenium ¹⁸⁶, rhenium ¹⁸⁸, ⁷⁵Selenium, ³⁵Sulphur, technetium ^99mAnd yttrium ⁹⁰In some embodiments, ¹²⁵I normally preferably uses, technetium ^99mAnd indium ¹¹¹Also normally preferred, because their energy are low and be suitable for the detection of wide scope.

Can produce radiolabeled albumen of the present invention or peptide according to method well known in the art.For example, they can by with sodium iodide or potassiumiodide and chemical oxidizing agent clorox for example, perhaps for example lactoperoxidase contact and carry out iodate of enzymatic oxidn agent.Can pass through the ligand exchange method, use technetium ^99mMark albumen of the present invention or peptide, for example: use solution of tin reduction pertechnetate (pertechnate), reductive technetium chelating is applied to this post to the Sephadex post and with peptide; Perhaps, for example pass through for example SNCl of incubation pertechnetate, reductive agent by direct labeling technique ₂, damping fluid for example sodium phthalate-potassium solution and peptide.The intermediate functional group that is generally used for being bonded to the radio isotope that metal ion exists peptide is diethylene triamine pentacetic acid (DTPA) (DTPA) and edathamil (EDTA).Also consider the use fluorescent marker, comprise rhodamine, fluorescein isothiocyanate and renographin.

In some embodiments, claimed albumen or peptide can be connected to second binding partner or be connected to the enzyme (enzyme label) that can produce coloured product after chromophoric substrate contacts.The example of suitable enzyme comprises urase, alkaline phosphatase, (horseradish) catalase and glucose oxidase.Preferred second binding partner is vitamin H and avidin or Streptavidin compound.The use of this type of marker is well known to those skilled in the art, and is described in for example United States Patent (USP) 3,817,837,3,850,752,3,939,350,3,996,345,4,277,437,4,275,149 and 4,366,241, incorporate this paper all by reference into for every piece.

In other embodiments, targeting moiety can operationally be coupled to nano particle.Nano particle includes but not limited to Radioactive colloidal gold and silver nano-grain.Metal nanoparticle demonstrates color in the visible spectrum zone.Believe that these colors are results that excite of surface plasma resonance in the metallic particles, and these colors for the insulativity of particulate size, shape and state of aggregation, surrounding medium, ion the absorption of this particle surface be extremely responsive (referring to, for example U.S. Patent application 20040023415, incorporate this paper by reference into).

J. linking agent

Linking agent also can be included in the fusion rotein or other construct of the peptide that comprises target CRKL; For example, linking agent can be used for the target peptide is conjugated to liposome or therapeutic compound.Difunctional crosslinking agents has been widely used in multiple purpose, comprises preparation affinity matrix, the binding site and the structural research of modifying and stablizing each class formation, evaluation part and acceptor.The same difunctionality reagent that carries two same functional group be proved to be induction phase highly effectively with and different macromole or macromolecular subunit between crosslinked, and polypeptide ligand is connected to their specific binding site.Heterobifunctional reagent contains two different functional groups.By utilizing the differential responses activity of two different functional groups, can be optionally and regularly control crosslinked.Can classify to it according to the specificity of bifunctional cross-linker's functional group, for example, amino, sulfydryl, guanidine radicals, indoles, carboxyl specificity group.In these, the reagent that relates to free amine group is welcome especially, and this is because their commercial availability, synthetic easiness and the reaction conditions of using them are gentle.Most Heterobifunctional linking agents contain primary amine reaction group and thiol-reactive group.

The illustrative methods that is used for crosslinking ligand and liposome is described in United States Patent (USP) 5,603, and 872 and 5,401,511, incorporate this paper all especially by reference in full into for every piece.Can multiple part be covalently bond to surface of liposome by the crosslinked of amine residue.Prepared liposome by sophisticated program, particularly multilamellar vesicle (MLV) or unilamellar vesicle, for example microemulsified liposome (MEL) and big unilamellar liposome (LUVET), each contains phosphatidylethanolamine (PE).Adding PE in liposome provides active function residue primary amine, and it is positioned at surface of liposome, is used for crosslinked purpose.Successfully will be connected to the PE-liposome such as the part of epidermal growth factor (EGF).Part covalently is bonded to the discontinuous site on the surface of liposome.The number in these sites and surface density are by the type decided of the prescription and the liposome of liposome.Surface of liposome can also have and is used for non-covalent bonded site.In order to form the covalent conjugates of part and liposome, linking agent is studied with regard to validity and biocompatibility.Linking agent comprises glutaraldehyde (GAD), difunctional epoxide ethane (OXR), ethylene glycol diglycidylether (EGDE) and water-soluble carbodiimide, preferred 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).By the chemistry of complex of linking agent, the amine residue of recognition material and being connected of liposome have been set up.

The method (United States Patent (USP) 5,889,155 is incorporated this paper especially by reference in full into) of Heterobifunctional linking agent and use linking agent has been described in another example.Linking agent is the combination of nucleophilic hydrazides residue and electrophilic maleimide residue, allows for example coupling of aldehyde and free mercaptan.Can modify with crosslinked different functional group linking agent.

Nucleic acid

According to nucleic acid of the present invention can encode target peptide, targeting antibodies, therapeutical peptide, fusion rotein or other albumen or peptide.Nucleic acid can be derived from genomic dna, complementary DNA (cDNA) or synthetic DNA.

" nucleic acid " used herein comprises strand and molecule two strands, and the nucleic acid and the nucleic acid analog of DNA, RNA, chemically modified.Consider that the nucleic acid in the scope of the invention can be almost big or small arbitrarily, depend in part on the length of encoded protein or peptide.

Consider that target peptide and fusion rotein can be by any nucleic acid sequence encodings of the suitable aminoacid sequence of coding.The password sublist of use standard, the nucleic acid of the aminoacid sequence that design and generation coding are wanted is well known to those skilled in the art.In preferred embodiment, can change selected each amino acid whose codon that is used to encode, so that the expression optimization of target host cell amplifying nucleic acid.

The targeted of gene therapy vector

In some embodiments, the target peptide can be coupled to gene therapy vector, so that selectivity or preferential target are at the cell of cell surface expression CRKL, for example some tumour cells.Exist a plurality of can be with the method for gene therapy vector transfered cell.In some embodiments of the present invention, described gene therapy vector comprises virus.The ability of some viruses " enter cell, be integrated into the host cell gene group or keep free and stable and express virogene effectively by receptor-mediated endocytosis " becomes it alien gene is shifted good candidate (Ridgeway, 1988 that enter mammalian cell; Nicolas and Rubinstein, 1988.; Baichwal and Sugden, 1986; Temin, 1986).Preferred gene therapy vector generally is a virus vector.Dna virus as gene therapy vector comprises papovavirus (for example, simian virus 40, bovine papilloma virus and polyomavirus) (Ridgeway, 1988; Baichwal andSugden, 1986) and adenovirus (Ridgeway, 1988; Baichwal and Sugden, 1986).

Preferably be used for one of the method for sending in the body and comprise the use adenovirus expression carrier.Though it is lower that known adenovirus carrier is integrated into the ability of genomic dna, this feature is remedied by the high efficiency gene transfer that these carriers provide." adenovirus expression carrier " includes but not limited to such construct, and described construct comprises and is enough to (a) and supports the packing of construct and (b) be expressed in wherein the antisense of being cloned or the adenoviral sequence of just polynucleotide.

Adenovirus carrier has been used for expression (people such as Levrero, 1991 of eukaryotic gene; People such as Gomez-Foix, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991).Comprise tracheal instillation (people such as Rosenfeld, 1991 about the research that recombinant adenovirus is applied to different tissues; People such as Rosenfeld, 1992), injection (Herz and Gerard, 1993) and three-dimensional the location are seeded to brain people such as (, 1993) Le Gal La Salle in intramuscular injection people such as (, 1993) Ragot, peripheral vein.

In preferred embodiment, can be coupled to the peptide (its target is at surface expression and carry the cell of CRKL, for example tumour cell) of target tumor and obtain some benefits by treating molecule or material.Particularly, will go back to the nest to the part of tumor vascular system and be coupled to cytotoxic drug or short apoptosis peptide, to be created in the experiment mice model that carries tumor xenogeneic graft more effectively and the lower compound of toxicity (people such as Arap, 1998 with respect to parent compound; People such as Ellerby, 1999).The RGD-4C peptide is inserted in the surface protein of adenovirus and has produced adenovirus carrier, it can be used for the gene therapy (people such as Arap, 1998) of target tumor.

By for example comprising targeting moiety of the present invention, and/or at the part of cell surface binding site, peptide motif allows to carry out cell-targeting.Peptide motif is optional can to comprise other element that is used for cell-targeting (for example, single-chain antibody sequence).Can produce peptide binding motif by the mode of inserting, it can comprise for example natural and the non-natural sequence, perhaps is made up of the non-natural sequence fully.Owing in chimeric fiber albumen, insert peptide motif that the alpha-non-natural amino acid sequence produces can be high-affinity peptide (promptly, when providing with low relatively concentration, with its homology binding site CRKL bonded peptide for example), or the peptide of low affinity (promptly, when providing, with its homology binding site CRKL bonded peptide for example) with high relatively concentration.But preferably, the peptide motif that is produced is the motif of high-affinity, and particularly those have the peptide motif of high-affinity to its homology binding site owing to the restriction in adenoviral fiber protein.

Other gene transfer vector can be from retroviral construct (Coffin, 1990).In order to make up retroviral vector, replace some virus sequence in the nucleic acid insertion viral genome with the coding target protein, to produce the virus of self-replacation defective type.In order to produce virus, made up and comprise gag, pol and env gene but do not contain LTR and the assembling clone of assembling component people such as (, 1983) Mann.When the recombinant plasmid that comprises cDNA and retrovirus LTR and assembling sequence is imported this clone (by for example calcium phosphate precipitation), the assembling sequence allows the rna transcription thing of recombinant plasmid to be fitted into virion, it is secreted into (Nicolas and Rubenstein, 1988 in the substratum then; Temin, 1986; People such as Mann, 1983).Collect the substratum that contains recombinant retrovirus then, optional concentrating, and be used for transgenosis.Retroviral vector can infect the various kinds of cell type.But, integrate and stably express needs division people such as (, 1975) Paskind of host cell.

Other virus vector can be used as the target gene therapy carrier.Can use derived from virus vaccinia virus (Ridgeway, 1988 for example; Baichwal and Sugden, 1986), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonatand Muzycska, 1984) and the carrier of simplexvirus.

In further embodiment of the present invention, the gene therapy construct can be assemblied in the liposome.The liposome-mediated delivery of nucleic acids and the vivoexpression of foreign DNA are quite successful.People such as Wong (1980) have proved the feasibility that liposome-mediated foreign DNA is sent and expressed in cultured chick embryo tire, HeLa and hepatocellular carcinoma cells.People such as Nicolau (1987) have successfully carried out the liposome-mediated transgenosis in the intravenous injection rat afterwards.

Gene therapy vector of the present invention can comprise multiple transgenosis, and it is the coding DNA or the RNA of expression vector normally.Gene therapy can be used for the expression of expression, VEGFR-1/NRP-1 of therapeutic genes to strengthen angiogenesis or to be used to suppress the expression of VEGFR-1/NRP-1, with treatment and angiogenesis disease states associated.The form of DNA can be the derivative of a part, the genetic stocks derived from virus, linear DNA, carrier (P1, PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequences, recombinant DNA, chromosomal DNA, oligonucleotide, antisense DNA or these groups of DNA, plasmid DNA, the plasmid DNA of cDNA, polymerization in vitro.The form of RNA can be the derivative of RNA, recombinant RNA, chimeric sequences, sense-rna, siRNA (siRNA), ribozyme or these groups of oligonucleotide RNA, tRNA (transfer RNA), snRNA (little nRNA), rRNA (ribosome-RNA(rRNA)), mRNA (messenger RNA(mRNA)), polymerization in vitro.Antisense polynucleotides is the polynucleotide that disturb the function of DNA and/or RNA.Antisense polynucleotides includes but not limited to: morpholinyl oligonucleotide (morpholinos), 2 '-O-methyl polynucleotide, DNA, RNA etc.SiRNA comprises duplex structure, contains 15-50 base pair usually, preferred 21-25 base pair; And have with the target gene of intracellular expression or RNA is identical or much at one nucleotide sequence.Interference may cause the inhibition expressed.In addition, DNA and RNA can be strand, two strands, three chains or four chains.

Pharmaceutical composition

Pharmaceutical composition of the present invention comprise significant quantity one or more described target tumors peptide or other be dissolved in or be scattered in reagent in the pharmaceutically acceptable carrier.Word " pharmaceutically or on the pharmacology acceptable carrier " is meant, when for example the people uses to animal (optionally), does not produce the molecular entity and the composition of disadvantageous, allergic or other untoward reaction.Comprising the peptide of at least a target tumor of the present invention or the preparation of drug combination of other activeconstituents is that those skilled in the art can be known according to the disclosure, as Remington ' sPharmaceutical Sciences, 18th Ed.Mack Printing Company, illustrated in 1990, it incorporates this paper by reference into.In addition, for to the using of animal (for example people), it should be understood that preparation should satisfy the regulation of the desired sterility of FDA biological standard office (Office ofBiological Standards), pyrogenicity, Generally Recognized as safe and purity rubric.

" pharmaceutically acceptable carrier " used herein comprises arbitrarily and all solvents, dispersion medium, encrusting substance, tensio-active agent, antioxidant, sanitas (antibacterial agent for example, anti-mycotic agent), isotonic agent, the absorption delayed-action activator, salt, sanitas, medicine, the medicine stablizer, gel, tackiness agent, vehicle, disintegrating agent, lubricant, sweeting agent, flavouring agent, material and combinations thereof such as dyestuff, as known to persons of ordinary skill in the art (referring to, Remington ' sPharmaceutical Sciences for example, the 18th edition, Mack Printing Company, 1990, the 1289-1329 page or leaf is incorporated this paper by reference into).Only with the inconsistent any conventional carrier of activeconstituents, otherwise consider that it is used for pharmaceutical composition.

Composition can comprise dissimilar carriers, and this depends on whether it uses with the form of solid, liquid or aerosol, with and whether need be aseptic to be used for the route of administration such as injection.The present invention is administration in the following manner: intravenously, intracutaneous, transdermal, in the sheath, intra-arterial, intraperitoneal, in the nose, intravaginal, internal rectum, the surface, intramuscular, subcutaneous, mucous membrane, mouthful, the surface, local, suck (for example aerosol suction), injection, infusion, continue infusion, direct targeted cells is bathed by regional perfusion, pass through conduit, by lavation, form with frost, form (for example liposome) with oil/fat composition, or by any other method or aforesaid arbitrary combination, as known to persons of ordinary skill in the art (referring to, Remington:TheScience and Practice of Pharmacy for example, the 21st edition, Mack Printing Company, 2005, incorporate this paper by reference into).

In addition, according to the present invention, in the pharmaceutically acceptable carrier that contains or do not contain inert diluent, provide the composition of the present invention that is fit to use.Described carrier should be absorbable, and comprises that liquid, semisolid promptly stick with paste, or solid carrier.Only to the recipient or to the deleterious any conventional media of treatment validity, reagent, the diluent or carrier of the composition that wherein comprises, otherwise its application at the composition of using that is used for implementing method of the present invention is suitable.The example of carrier or thinner comprises fat, oil, water, salts solution, lipid, liposome, resin, tackiness agent, weighting agent etc., or its combination.Composition can also comprise multiple antioxidant to stop one or more compositions oxidized.In addition, sanitas can stop action of microorganisms, and described sanitas is for example antibacterium and anti-mycotic agent, includes but not limited to Tegosept E (for example Tegosept M, propylben), butylene-chlorohydrin, phenol, Sorbic Acid, Thiomersalate or its combination.

According to the present invention, composition mixes in the mode of any routine and practicality with carrier, that is, and and by solution, suspension, emulsification, mixing, encapsulation, absorption etc.This class method is the conventional uses of those skilled in the art.

Combined therapy

For the validity of the peptide that increases the isolating target tumor that comprises the CRKL binding motif, may need these compositions and other reagent or for example antitumor and anticancer agent associating of methods of treatment." anticancer " reagent can negatively influence the cancer among the experimenter, for example, the apoptosis by kill cancer cell, inducing cancer cell, reduce cancer cells growth velocity, reduce metastasis rate or number of times, reduce the tumour size, suppress tumor growth, reduce blood supply, promote immunne response, prevention or suppress the progress of tumour or increase life-span of cancered experimenter at cancer cells or tumour to tumour or cancer cells.More generally, will provide these other compositions with the combined amount that is enough to cell killing or suppresses its propagation.This method can comprise cell is contacted with reagent or a plurality of factor simultaneously with expression construct.This can realize like this: cell with single composition or comprise that the pharmacological preparation of two kinds of reagent contacts, is perhaps contacted cell with two kinds of different compositions or preparation simultaneously, and wherein a kind of composition comprises expression construct, and another kind comprises second reagent.

Tumour cell is the subject matter that Clinical Oncology runs into to the resistance of chemotherapy and radiation reagent.One of target of present cancer research is to find by chemotherapy and radiation is combined with gene therapy to improve the mode of its effect.For example, when hsv thymidine kinase (HS-tK) when gene is transported to cerebral tumor, successfully having been induced the susceptibility (Culver waits the people, 1992) of resisting viral reagent ganciclovir by the retroviral vector system.In the context of the present invention, consider except other short apoptosis or Cycle Regulation agent that the peptide of target tumor can be united use with chemotherapy, radiotherapy or immunotherapy similarly.

Perhaps, can before or after other reagent is administered, carry out gene therapy, be spaced apart several minutes to several weeks.Using in the embodiment of other reagent and expression construct to cell respectively, people will guarantee that generally the effective time period can not lose efficacy between each time of carrying, thereby described reagent and expression vector will be brought into play favourable combined effect in cell.Under these circumstances, consider, can be in the about 12-24 of each interval hour, more preferably in the about 6-12 of each interval hour cell is contacted with two kinds of application methods.In some cases, may need the time period of significant prolongation treatment, still, interval a couple of days (2,3,4,5,6 or 7) are to several weeks (1,2,3,4,5,6,7 or 8) between each time administration.

Can use multiple combination, the peptide of target tumor is " A ", and second reagent for example radiotherapy or chemotherapy is " B ":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Use therapeutic expression construct of the present invention to the patient and will consider the toxicity (if any) of carrier according to the general procedure that is used to use chemotherapeutics.Expect if necessary, with the repetitive therapy cycle.Also consider and to treat and surgical intervention with described excess proliferative cell therapy combined utilization multiple standards.

A. chemotherapy

Cancer therapy also comprises treatment multiple and based on therapeutic combination chemistry and that radiate.Combined therapy comprises, for example, cis-platinum (CDDP), carboplatin, procarbazine, mustargen, endoxan, camptothecine, ifosfamide, melphalan, Chlorambucil, busulfan, nitrosourea, gengshengmeisu, daunorubicin, Zorubicin, bleomycin, Plicamycin, mitomycin, Etoposide (VP16), tamoxifen, raloxifene, the estrogen receptor wedding agent, taxol, gemcitabine, nvelbine, farnesyl protein transferase inhibitors, trans platinum, 5 FU 5 fluorouracil, vincristine(VCR), vinealeucoblastine(VLB) and methotrexate, or the aforementioned every any analogue or the variant of deriving.

B. radiotherapy

Cause that dna damage and the other factors that has been widely used comprise, be commonly called gamma-radiation, X-ray and/or the radio isotope orientation is delivered to tumour cell.Also consider the dna damage factor of other form, for example microwave and UV-radiation.Most possible is, all of these factors taken together is for DNA, for the precursor of DNA, duplicating and repairing and for chromosomal assembling with keep, have multiple damaging effect for DNA.The dosage range of X-ray is: 50-200 roentgen's dosage every day (for the time period (3 to 4 week) that prolongs) is to 2000-6000 roentgen's single dose.Radioisotopic dosage range changes very greatly, and it depends on the intensity and the type of the ray of isotopic transformation period, emission, and the picked-up situation of tumour cell.

When being applied to cell, term " contact " and " being exposed to " are used for describing the process of " therapeutic construct and chemotherapy or radiotherapy reagent are delivered to target cell or make it and target cell is directly adjoined " in this article.In order to realize that cell kills or suppresses, according to effective cell killing or stop its splitted number of combinations that two kinds of reagent are delivered to cell.

C. immunotherapy

Generally speaking, immunotherapy depends on and uses immunological effect daughter cell and molecule with target and destruction of cancer cells.Described immune effector can be for example for the specific antibody of some marks on the tumor cell surface.Described antibody can be used alone as the treatment effector, and perhaps it can be raised other cell and kills to carry out actual cell.Antibody can also be conjugated to medicine or toxin (chemotherapy, radionuclide, ricin A chain, Toxins,exo-, cholera, Toxins, pertussis etc.) and only be used as targeting agent.Perhaps, effector can be to carry directly or indirectly and the lymphocyte of the interactional surface molecular of tumour cell target.Various effector cells comprise: cytotoxic T cell and NK cell.

Therefore, immunotherapy can be used as the part of combined therapy, with the treatment associating of the peptide that uses target tumor.The general method of combined therapy is discussed hereinafter.Usually, tumour cell must carry some and be easy to by the mark of target,, is not present in the mark on most other cells that is.Have a lot of tumor markerses, and any item of these marks may be suitable for the target of the context of the invention.Common tumor markers comprises carcinomebryonic antigen, prostate specific antigen, urologic neoplasms related antigen, embryonal antigen, tyrosine oxidase (p97), gp68, TAG-72, HMFG, sialylated Louis's antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.

D. gene

In another embodiment, second treatment is gene therapy, wherein before the first treatment reagent of the peptide that comprises the isolating target tumor that contains the CRKL binding motif, administering therapeutic polynucleotide simultaneously afterwards or with it.Second carrier of therapeutic agent and coding one of following gene product unite the anti--hyper-proliferative effect of sending with having to the combination of target tissue.

E. operation

About 60% patient who suffers from cancer will accept the operation of some type, comprise operation preventative, diagnostic or property by stages, radical-ability and taking stopgap measures property.The operation of radical-ability is to treat the cancer therapy that (treatment for example of the present invention, chemotherapy, radiotherapy, hormonotherapy, gene therapy, immunotherapy and/or surrogate therapeutic) unites use with other.

The operation of radical-ability comprises surgical blanking, and wherein all or part of cancerous tissue is removed, excised and/or destroy by physics.Tumor resection is meant that physics removes at least a portion tumour.Except tumor resection, the treatment of being undertaken by operation comprises the operation (mohs' technique) that laser surgey, cryosurgery, electrosurgery and microscope are controlled.Consider that also the present invention can unite use with the healthy tissues that shallow property cancer, precancer or accidental quantity are shown in removal.

Excise after part or all cancerous cells, tissue or the tumour, may form the chamber in vivo.Can finish treatment by perfusion, direct injection or with other anticancer therapy topical application to this zone.This type of treatment can repeat, and for example per 1,2,3,4,5,6 or 7 day, or per 1,2,3,4 and 5 weeks, or per 1,2,3,4,5,6,7,8,9,10,11 or 12 months.These treatments also can be different dosage.

F. other reagent

Consider that other reagent can unite use to improve the therapeutic effect of treatment with the present invention.These other reagent comprise, the inhibitor of the reagent of immunomodulator, the reagent of going up mediation GAP juncture that influences cell surface receptor, inhibition cell and differentiation agents, cell adhesion, or the reagent of the susceptibility of the cell pair cell inducer of apoptosis of increase hyper-proliferative.Immunomodulator comprises tumour necrosis factor; Interferon alpha, β, γ; IL-2 and other cytokine; F42K and the similar thing of other cytokine; Or MIP-1, MIP-1 β, MCP-1, RANTES and other chemokine.Also consider cell surface receptor or its part for example the rise of Fas/Fas part, DR4 or DR5/TRAIL will strengthen apoptosis-inducing ability of the present invention for the autocrine or the paracrine effect of excessive proliferated cell by setting up.Strengthen intercellular signal by the number that increases the GAP juncture and conduct the anti--hyper-proliferative effect that will strengthen the cell colony of contiguous hyper-proliferative.In other embodiments, reagent and the differentiation agents that suppresses cell can be united use to improve the anti--hyper-proliferative effect of treatment with the present invention.The inhibitor of considering cell adhesion can improve effect of the present invention.The example of the inhibitor of cell adhesion is focal adhesion kinase (FAK) inhibitor and lovastatin.Also consider other reagent of the susceptibility of the cell pair cell apoptosis that increases hyper-proliferative, for example antibody c225 can unite use to improve therapeutic efficiency with the present invention.

Hormonotherapy also can be used in combination with the present invention's associating or with above-described any other cancer therapy.Hormone can be used for treating some cancer, and for example mammary cancer, prostate cancer, ovarian cancer or cervical cancer are to reduce some hormone for example testosterone or estrogenic level or block its effect.This treatment is united as the treatment selection or in order to reduce the risk of transfer with at least a other cancer therapy usually.

Embodiment

Comprised that following examples are with further explanation all respects of the present invention.Those skilled in the art will recognize that disclosed technology is represented effect good technical and/or the composition when enforcement is of the present invention that the inventor finds in following examples, therefore, can consider that it constitutes preference pattern of the invention process.But those skilled in the art will recognize according to the disclosure, not break away from the spirit and scope of the present invention, can much change for disclosed embodiment, and still obtain same or analogous result.

Select the tumor-homing peptide in embodiment 1 body

Show random peptide library (people such as Arap, 1998 to nu/nu (naked) the mouse intravenously application of bacteriophage of carrier DU145-deutero-prostate cancer heterograft; Pasqualini andRuoslahti, 1996; People such as Pasqualini, 2001), after 24 hour cycle period, reclaim tumour.After the process three-wheel is selected, reclaim the enrichment colony of the phage of target tumor, will check order corresponding to the sub DNA of peptide insertion that each phage clone shows by (table 1).Select the peptide of dominance to carry out the function sign, its aminoacid sequence is YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1).At first, in the mouse of carrying DU145-deutero-tumour, assess the cancer target specificity of YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) peptide in the body.Use through the general intravenously after the phage clone of single demonstration YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1), observe significantly to the going back to the nest of tumor xenogeneic graft (do not have insert son phage as negative control), in several contrast organs, do not see or almost do not have a detectable phage location.Consistent therewith, by using water and organic phase separation determination method (people such as Giordano, 2001) with based on the immunofluorescence assay (KS1767 cell) of phage, at external same target the DU145 prostate cancer cell, and the phage of finding show peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) and combining of tumor cell surface be far longer than show peptide not insert sub negative control phage and its combine (Figure 1A, B).

Table 1

Next, assessed the internalization ability of selected peptide.For this reason, with short apoptosis peptide (people such as Arap, 2004 of selectively targeted eukaryotic cell plastosome film; People such as Javadpour, 1996; People such as Ellerby, 1999; People such as Kolonin, 2004; People such as Zurita, 2004) merge to the tumor-homing peptide.Kill the internalization that (Fig. 1 C) shows that peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) mediation part instructs with respect to the cell of the target that contrasts.Confirmed apoptosis (Fig. 1 D) by annexin-V dyeing assay method.It should be noted that short apoptosis peptide the selectivity target and in turn to the target module based on the antineoplaston of intending peptide (people such as Arap, 2004; People such as Ellerby, 1999; People such as Kolonin, 2004; People such as Zurita, 2004) design provides possibility.To sum up, these results show that peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) target tumor cell also can internalization.

Reagent

All clone all from U.S. typical cells storehouse (American Tissue TypeCollection, ATCC).Use following antibody in the research described herein: anti--CRKL (Santa Cruz, Cell Signaling, Epitomics or Upstate Biotechnology), anti--phosphorylation-CRKL (Cell Signaling), anti-β ₁Integrin (Chemicon or BDTransduction Laboratories), anti--IL11R (Santa Cruz), anti--β ₃With anti--β ₅Integrin 47, anti-EGFR 48, anti--grb2 (Santa Cruz), anti--α 6 integrins (Chemicon), anti--AHSG/Feutin A (R﹠amp; D Systems), pre-immune serum (Jackson Laboratory), anti--FAK (Upstate), anti--histone h1 (Santa Cruz), anti--phosphorylation-paxillin (Cell Signaling), anti--phosphorylation-130Cas (Santa Cruz), anti--phosphorylation-Erk1/2 (Cell Signaling or Biosource), anti--phosphorylation-Elk-1 (CellSignaling), anti--His (Santa Cruz), anti--gst (Santa Cruz) and resist-GAPDH (Ambion).Specification sheets (AnaSpec) according to us synthesizes peptide and carries out cyclisation.The male nude mouse in 6 ages in week obtains (Harlen) by the commercial channel, according to people such as Arap, and 2004; People such as Marchio, 2004 description prepares tumor xenogeneic graft.The mechanism's the care of animal of Anderson tumor center of University of Texas (UTMDACC) and the use council (IACUC) examine and have ratified all experimental arrangements.

Phage shows the selection of random peptide library

According to describing (people such as Arap, 1998; People such as Arap, 2004; Pasqualini andRuoslahti, 1996; People such as Pasqualini, 2001) carry out phage selection in the body.Nude mouse systemic administration (tail vein) to the tumor xenogeneic graft that carries derived from human DU145 prostate cancer cell shows to have general arrangement X ₂CX ₁₂CX ₂(C, halfcystine; X, arbitrarily residue) the random phage library of insertion, make its circulation 24 hours.Mouse is carried out deep anaesthesia, the tumor resection heterograft, weigh, reclaim and handle bonded phage colony (people such as Arap, 1998; People such as Arap, 2004; Pasqualini and Ruoslahti, 1996; People such as Pasqualini, 2001).Carry out interior selection of body of 3 continuous rounds.

Embodiment 2 tumor-homing peptide sequences are similar to the integrin extracellular domain of modulability

In order to determine that whether the peptide sequence of identifying is similar to native protein, carries out the similarity searching of YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) and other selected peptide sequence.By using standard blast search, carrying out the protein sequence comparison then, find that peptide that the phage of all uniquenesses shows all is similar to and be present in β at online database ₁Sequence on the integrin.Unexpected is peptide sequence YRCTLNSPFFWEDMTHECHA of dominance (SEQ ID NO:1) and β ₁Clump shape albumen-brain signal albumen-integrin (PSI) extracellular domain (residue 26-78) of integrin chain has similarity (Fig. 2 A); Find that in addition other selected peptide also appears at same area (Fig. 2 C).Then, whether the similarity of having determined selected peptide sequence YRCTLNSPFFWEDMTHECHA (SEQ IDNO:1) is β ₁The PSI structural domain of integrin sequence is specific, is common for other known integrin β chain perhaps.Carry out reaching a conclusion after Fitting Analysis and the molecular simulation: YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) and β ₁Sequence identity between the PSI structural domain of integrin is best comparison (Fig. 2 B) really.

Sequence alignment is analyzed

Analyze tumor-homing phage display peptide and β by using based on the peptide adapting software of writing among the Perl 5.8.1 of RELIC50 ₁Sequence alignment between the integrin.This program is based on predetermined residue window size, with from N to the C albumen end mode of residue displacement one by one, calculates the peptide sequence of affine selection and the similarity between the target protein sequence.Calculate the peptide-protein similar score of each residue based on BLOSUM62 amino-acid substitution matrix (making amendment) to consider the expression of rare amino acid.With threshold value setting is at least 4 identical residues between peptide and the albumen section, to distinguish the remarkable similarity of mating with non-specific background.

Embodiment 3 tenuigenin joint albumen are as the acceptor of PSI structural domain sample tumor-homing peptide

The active PSI structural domain that has characterized integrin well (people such as Shi, 2005 have just been regulated; People such as Mould, 2005; People such as Arnaout, 2005; People such as Juliano, 2004).As if according to selection result, the PSI structural domain also may be as β ₁Ligand-receptor binding site in the integrin plays a role.In view of this, use affinity chromatography to identify the binding partners of tumor-homing peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1).At first, clear up DU145 deutero-cell extract in advance by the control peptide post, the extract that will clear up in advance carries out acid wash-out then by tumor-homing YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) peptide post then.Detect from post elute corresponding to～proteic specificity the gel band of 40-KDa.

It is No. 10 conditioning agents of the proteic chicken tumor disease of kinases sample poison that mass spectrum and database analysis have disclosed the proteic identity of this gel band, registration number NP005198 (SEQ ID NO:22) people such as (, 1993) ten Hoeve (being called CRKL).Immunoblotting by anti--CRKL antibody and purifying protein has been verified these results.Next, the reorganization His-tag fusion protein (rCRKL) of CRKL and structural domain rCRKL-SH2, rCRKL-SH3 (N) and the rCRKL-SH3 (C) of 3 correspondences thereof have been designed and have made up, to be used in conjunction with measuring.Find that peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) preferably is bonded to rCRKL by these proteic 2 SH3 structural domains; In contrast, almost do not have or detect less than the combination by the SH2 structural domain and with combine (Fig. 3 A) of reference protein.When under similar experiment condition, testing, show that several phage clones of uncorrelated peptide sequence do not demonstrate combination.Also use directly derived from β ₁The natural PS I structural domain of integrin (sequence NSTFLQEGMPTSA (SEQ ID NO:23); Residue 50 to 62) synthetic peptide (it is overlapping in natural PS I zone with peptide sequence that phage show to be selected) carried out combining research (Fig. 2 A, C).Similarly, β ₁Integrin PSI-derived peptide NSTFLQEGMPTSA (SEQ IDNO:23) is bonded to rCRKL, rCRKL-SH3 (N) structural domain and rCRKL-SH3 (C) structural domain (Fig. 2 B).In addition, prepared annular and linear peptides sequence from the PSI structural domain, the annular disulfide linkage of finding to exist in the PSI structural domain is for optional with combining of CRKL (Fig. 3 C).At last, shown that SH3 (C) structural domain is suppressed (Fig. 3 D) with interaction between the tumor-homing peptide by corresponding synthetic peptide and the specificity of phage clone own.These find to show: tumor-homing peptide and β ₁The SH3 structural domain of the specific PSI structural domain of integrin analogue YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) target CRKL.

Affinity chromatography and mass spectrum

Peptide affinity column by EDC and DADPA fixing resin (Pierce) preparation standard.Preparation DU145 tumour cell extract also makes it at first by non-specific control peptide post, then by tumor-homing peptide post.Thoroughly washing column uses glycine (pH 2.2) wash-out then, analyzes by SDS-PAGE.Then, gel is carried out coomassie dyeing.Detect～band of 40KDa, it is scaled off, carry out protein sequencing by mass spectrum at UTMDACC Proteomic Core Facility.This albumen is accredited as CRKL.Carry out the affinity purification of CRKL from the serum-free condition substratum, and (the reorganization gst-tag fusion rotein of Pro-＞Ala) is confirmed for peptide or mutant control peptide to use expressing tumor to go back to the nest.The serum-free condition substratum (cultivating 48 hours) that concentrates about 200ml is to carry out affinity purification.Recombination fusion protein is coupled to the gst-resin bead and is loaded into post.Spissated serum-free condition substratum is added to the coupling post and be incubated overnight.After the washing several times, the CRKL of elution of bound is to carry out the Western engram analysis.Use anti--gst and anti--CRKL antibody to survey trace.

Phage combination and protein-protein are measured

Carry out the proteic phage of purifying in conjunction with mensuration according to describing people 2001 such as () Giordano.According to former description (people such as Cardo-Vila, 2003; Smith and Scott, 1993) with the recombinant protein bag by to microtiter well.In brief, at 4 ℃ 50 μ l albumen (1 μ g/ml is among the PBS) are fixed in the microtiter well and spend the night.Use PBS with hole washing 2 times, use the PBS that contains 3%BSA, use the following phage among the 50 μ lPBS (containing 1.5%BSA) to carry out incubation: 10 room temperature sealing 2 hours ⁹T.U. wild-type tumor-homing phage (YRCTLNSPFFWEDMTHE CHA; SE Q ID NO:1), messy phage (YRFCTSPFHEWHLENTDMCA (SEQ ID NO:26), YRECTDSPHEFHLWNTMCAF (SEQ ID NO:27), YRCETDSPHEFHLWNTMCAF (SEQ ID NO:29), YRCETDSPHEFHLWNTFCAM (SEQ ID NO:30)), sudden change phage (YRCTLNSAFFWEDMTHECHA (SEQ ID NO:25), YRCTLNSPAAAEDMTHECHA (SEQ ID NO:28)), PSI deutero-ring-type phage (CNSTFLQEGMPTSAC; SEQ ID NO:23) or the fd-tet phage.After room temperature is carried out 1 hour, use PBS with hole washing 10 times, reclaim phage by infectation of bacteria.Use polyclone anti--ELISA that CRKL carries out confirmed the existence and the concentration of CRKL recombinant protein in the hole.Combine with containing the proteic of SH3 in order to detect the tumor-homing phage, use 250 η g/ml reorganization gst-SH3 structural domain (CRKL-D1, CRKL-D2, grb2-D1, grb2-D2, Lyn, src at 4 ℃; Pronomics), rCRKL albumen and negative control gst or BSA bag is spent the night by microtiter well.In conjunction with research, bag is by the His-label of 2 μ g/ml reorganization wild-type SH3-C and mutant SH3 (C) structural domain for SH3 (C) mutant.In each hole, add tumor-homing phage (10 ¹⁰T.U.) or fd-tet phage (do not have insert son), and according to description above carry out combination mensuration.Carried out CRKL and integrin β at bag in the hole of α 5 β 1, the α v β 3 in 1 μ g/ hole or α v β 5 integrins (Chemicon) ₁Between protein-protein interaction experiment.In order to assess gst-PSI for CRKL and β ₁The bonded of integrin suppresses, and gst-PSI that CRKL and concentration is cumulative or gst join in the hole of wrapping quilt then room temperature preincubation 15 minutes.After 3 hours, anti-by using-CRKL antibody, use anti--rabbit IgG that HRP-puts together to detect combining of CRKL and integrin then.For the integrin of confirming same amount is bonded to flat board, use the anti-integrin antibody (Amersham Pharmacia) of dilution in 1: 1500 to carry out parallel laboratory test.

Design and the structure of messy and mutant tumor-homing phage

In order to produce the phage clone that is used to study the proteic binding characteristic of CRKL, the phage that shows messy peptide sequence and mutant (Pro-＞Ala and Phe-Phe-Trp-＞Ala-Ala-Ala) have been designed and have made up from selected tumor-homing phage display peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) in conjunction with CRKL.With messy peptide sequence (YRFCTSPFHEWHLENTDMCA (SEQ ID NO:26), YRECTDSPHEFHLWNTMCAF (SEQ ID NO:27), YRCETDSPHEFHLWNTMCAF (SEQ ID NO:29), YRCETDSPHEFHLWNTFCAM (SEQ ID NO:30)), mutant (YRCTLNSAFFWEDMTHECHA (SEQ ID NO:25) and YRCTLNSPAAAEDMTHECHA (SEQ ID NO:28)) or natural PS I deutero-phage (CNSTFLQEGMPTSAC; SEQ ID NO:23) clone enters the fUSE5 carrier (Smith and Scott, 1993) of SfiI-digestion.In brief, use primer sets 5 ' GTGAGCCGGCTGCCC 3 ' (SEQ ID NO:68) and 5 ' TTCGGCCCCAGCGGC 3 ' (SEQ ID NO:69) (Sigma-Genosys) with the Taq-DNA polysaccharase (Promega) (volume is 20 μ l) of 2.5U, by pcr amplification 500 η g are converted into double-stranded DNA corresponding to every kind of synthetic oligonucleotide template (Sigma-Genosys) of the peptide that shows, amplification program is: 94 ℃, and 2 minutes; 35 circulations then: 94 ℃, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 30 seconds; 72 ℃ then, 5 minutes.Use the QIAquick neucleic acid to eliminate test kit (Qiagen) purifying and inserting the double chain DNA sequence that sub-flanking region comprises the BglI restriction site, and wash-out.37 ℃ use BglI with oligonucleotide digestion 2 hours, again purifying, and connect the fUSE5 carrier that enters SfiI-digestion.Produce phage clone to verify correct insertion and nucleotide sequence by pcr amplification.Test each phage clone in phage in conjunction with in measuring.

Peptide combination and internalization are measured

With synthetic tumor-homing peptide (YRCTLNSPFFWEDMTHECHA; SEQ IDNO:1) or PSI deutero-peptide (NSTFLQEGMPTSA; SEQ ID NO:23) the bag quilt seals then and washs to microtiter plate.In the flat board of bag quilt, add following reorganization His-label protein: rCRKL, rCRKL-SH2 structural domain, rCRKL-SH3 (N) structural domain and rCRKL-SH3 (C) structural domain.Uncorrelated reference protein (α 2-Heremans-Schmid glycoprotein; AHSG) as negative control.With mixture incubation, washing and use suitable antibody to carry out mark.Adding is conjugated to two of horseradish peroxidase (HRP) and resists, and adds tmb substrate (Calbiochem) then, and analyzes by automatization ELISA reader (Bio-Tek).In order to determine the inhibition activity of tumor-homing peptide, with PSI deutero-peptide or the phage clone that shows corresponding tumor-homing peptide with rCRKL-SH3 (C) structural domain incubation.Incoherent cyclic peptide sequence and nothing are inserted the phage (fd-tet) of son as negative control.With the mixture incubation and join in the hole through tumor-homing peptide bag quilt.After the incubation, washing hole uses suitable antibody to carry out mark, and handles according to description above.

According to describing (people such as Arap, 2004; People such as Ellerby, 1999; People such as Kolonin, 2004; People such as Zurita, 2004) measure via the glycylglycine bridge and merge to the internalization ability of the tumor-homing peptide of short apoptosis sequence.The synthetic tumor-homing peptide YRCTLNSPFFWEDMTHECHAGG (SEQ ID NO:67) that puts together- _D(KLAKLAK) ₂Or the control peptide of non-target _D(KLAKLAK) ₂, and the peptide of volumetric molar concentration such as cumulative joined in the DU145 cell.Measure cell survival by WST-1 reagent (Roche), measure apoptosis (people such as Zurita, 2004 by annexin-V dyeing; People such as Cardo-Vila, 2003).For the Position Research of tumor-homing phage, with cell and 10 ⁹T.U. tumor-homing peptide (YRCTLNSPFFWEDMTHECHA; SEQ ID NO:1) or negative control (fd-tet) phage incubation 6 and 24 hours together.Use 20mM glycine washing hole to remove nonspecific cell surface bonded phage, use 4% Paraformaldehyde 96 to fix then.With the cell of non-penetratingization with rabbit anti--fd phage antibody (Sigma) is room temperature incubation 2 hours, then with anti--rabbit IgG antibody (JacksonImmunoResearch) incubation 1 hour of Cy3-mark.Reuse 4% Paraformaldehyde 96 fixed cell, and under the situation that has DAPI (Vector Laboratories) mounting, use the Olympus fluorescent microscope to obtain image.

Embodiment 4 and the interactional sign of CRKL

Other research shows, except with the motif that contains single Pro residue combines, the SH3 structural domain also combines (Mayer, 2001 with PXXP and non-PXXP motif; People such as Sicheri, 1997; People such as Kang, 2000; People such as Kato, 2000; People such as Xu, 1997).Because the sequence shown in the SEQID NO:1 does not contain any this type of known motif, so use site-directed mutagenesis to assess the binding characteristic of tumor-homing peptide.The result of these researchs shows: depend on Pro residue and the annular Cys-Cys disulphide bridges (Fig. 4 A) that exists in the tumor-homing peptide with combining of CRKL-SH3 (C) structural domain; Fd-tet does not have the phage that inserts son and incoherent synthetic cyclic peptide as negative control.Also carried out the mutation analysis of CRKL SH3 (C) structural domain, the result shows that calmodulin binding domain CaM is at residue Gly ₂₃₆With Trp ₂₇₇Between (Fig. 4 C, D).Show that in addition tumor-homing phage (SEQ ID NO:1) and PSI deutero-phage (SEQ ID NO:23) all combine (Fig. 4 B) with recombinant C RKL.In addition, by use affinity chromatography determined can be from DU145 serum-free condition substratum purifying CRKL, under identical experiment condition,, use the contrast post of the mutant forms preparation of tumor-homing peptide no longer to combine with CRKL in detectable level.Measure by the mutual co-immunoprecipitation that uses membrane component to carry out, determined CRKL and β ₁Integrin forms the cell surface mixture; On the contrary, the control antibodies that produces at uncorrelated transmembrane receptor (comprising anti--IL11 acceptor, anti--the EGF acceptor) or (anti--β at the antibody of other integrin generation ₃With anti--β ₅) do not demonstrate and CRKL or β ₁The combination of integrin.At last, proved that the PSI structural domain with the recombinant protein formal representation can suppress CRKL and β in the dose-dependently mode ₁Protein-protein interaction (IC between the integrin ₅₀=20 η m); Consistent with the result of co-immunoprecipitation research, the contrast integrin does not demonstrate in conjunction with (Fig. 5).

The structure of mutant

All primer sets are summarized in table 2.Opening code-reading frame and clone by pcr amplification total length CRKL cDNA (Invitrogen) enter pET28a (Novagen) expression vector.By the cDNA of pcr amplification corresponding to each SH structural domain, and the clone enters the Sac I and the Xho I restriction site of described carrier.Verify all constructs by restrictive diges-tion and dna sequencing, its conversion is entered BL21 (Stragene) bacterial isolates, go up purification of recombinant proteins at His-label post (Qiagen).By coomassie dyeing and use anti--CRKL and anti--His antibody is verified the recombinant protein of purifying by the Western engram analysis

Be created in the CRKL mutant that SH3 structural domain (C-terminal) is undergone mutation by PCR mutagenesis.Primer sets is designed to remove 60bp (Δ 1 and Δ 2) and 54bp (Δ 3) and keeps reading frame.For PSI structural domain construct, prepared the ring-type and the linear peptides form of PSI structural domain.With after the annealing of PSI oligonucleotide (table 2), after use EcoRI digests, use Nucleotide to remove test kit (QIAGEN) purifying double chain oligonucleotide, the clone enters the pGEX4T-1 carrier then.In order to show CRKL albumen oligomerization, reorganization gst-CRKL that concentration is cumulative and fixed CRKL albumen (His-label recombinant forms) or fixed BSA are incubated overnight at 4 ℃, wash then 3 times.By using antibody detection protein-protein-interacting at gst.

Table 2:PCR primer (SEQ ID NO:32-53)

Embodiment 5 molecular imagings show that CRKL can be positioned the cell outside

Consistent with the above evidence that presents, molecular imaging proof CRKL and β ₁Integrin is located altogether.Join together, these studies show that on cell surface, CRKL and β ₁Specific molecular between the integrin interacts.Because the tumor-homing peptide is bonded to the surface of DU145 prostate cancer cell, so studied the possibility of " CRKL may also be positioned the cytolemma outside ".The facs analysis of DU145 cell shown with respect to the warp of contrast anti--cell surface (Fig. 6 A) of CRKL antibody labeling.Also studies confirm that the cell surface marker situation by immunofluorescence dyeing and the co-focusing imaging that under non-penetratingization condition, carries out.Also under non-penetratingization condition, on the superstructure level, studied cell surface location situation (TEM by scanning and transmission electron microscope; Fig. 6 B).These classical imaging techniques have produced the cell surface marker situation (Fig. 6 B) of CRKL once more.Also used other two kinds of biochemical methods to confirm the cytolemma location of CRKL: the cell surface marker and the stain remover membrane sepn that are undertaken by biotinylation.By these two kinds of independent solutions, find that CRKL is present on the cytolemma.For these research, at the antibody of incoherent intracellular protein as negative control.To sum up, these results show: except well-known be present in the tenuigenin, CRKL albumen also is positioned cell surface.

Cell surface and film location

According to describing the cell surface marker that people such as (, 2004) Monferran uses vitamin H.In brief, use the non-perviousness vitamin H of film-LC-LC-NHS EZ link (Pierce) mark DU145 cell.Biotinylated membranin washing, dissolving and use Streptavidin pearl (Pharmacia) are made its clarification by immunoprecipitation.Suitable antibodies shown in the use is carried out the Western engram analysis.According to description, wash in a pan sieve and real-time analysis (BRASIL) method of the reaction part that filters out people such as (, 2001) Giordano is carried out the phage cell surface in conjunction with mensuration on the DU145 cell by biology.According to describing people such as (, 2003) Mintz, carry out membrane sepn.Suitable antibodies shown in the use is surveyed immunoblotting.On LSM 510 (Carl Zeiss) Laser Scanning Confocal Microscope, obtain the burnt image of copolymerization.The DU145 cell is grown on the slide of fibronectin bag quilt, and the Paraformaldehyde 96 (PFA) of use 4% is fixing and use suitable antibody (polyclone CRKL antibody and mono-clonal AHSG antibody) to carry out mark.Obtain electron microscope image at High Resolution ElectronMicroscopy Core Facility (JSM 5900 scanning and JEM 1010 transmission electron microscopes).Prepare gold nano grain antibody-conjugate by the gold that in Sodium Tetraborate, mixes 20-25 η m or 40-45 η m.Verify golden link coupled nano particle by tem analysis.Use suitable antibody (monoclonal anti-CRKL, anti--β on ice ₁Integrin and anti--AHSG antibody) mark DU145 cell, fluorescence two anti-marks by puting together then, and pass through facs analysis.

Embodiment 6 functional studies and potential CRKL transporting mechanism

Because CRKL does not have classical membrane spaning domain, whether come with assessment CRKL from tumor cell secretion so carried out research.The result shows that the DU145 cell of cultivating is the CRKL of the without phosphorus acidifying form of secretion really in serum free medium.In contrast, detecting in the control cells substratum less than CRKL: as use anti--CRKL or use shown in the immunoprecipitation that several control antibodies carry out.In order to assess the generality of these observationss, studied one group of tumor cell line in the serum free medium, and found that they also secrete without phosphorus acidifying CRKL (Fig. 7 A), therefore show: this phenomenon is not that cell category is specific.Next, carried out research and whether had any detectable effect for cell proliferation and migration with the secretion of determining CRKL.In order to prove specificity, in the serum free medium of DU145 cell culture, add anti--CRKL neutralizing antibody.The result shows, neutralize really extracellular CRKL and reduce cell proliferation (Fig. 7 B) and cell migration (Fig. 7 C) of anti--CRKL antibody; The serum of several control antibodies or pre-immunity does not produce the detected effect (Fig. 7 B, C) for cell proliferation or migration.In order further to understand the biological action of CRKL in the tumour cell environment, used the siRNA technology to strike low CRKL; The same discovery when the expression decreased of CRKL, causes that cell proliferation, adhesion and migration significantly reduce (Fig. 8 A-C).As other contrast, demonstration can be saved the minimizing (Fig. 8 D) of cell proliferation by external source CRKL, strikes in low cell or the serum free medium cultured cells at CRKL siRNA, only detects the apoptosis (less than 1%) of background level.Find that also strike CRKL in the low cell by siRNA and can reduce combining of tumor-homing phage and cell, this illustrates that tumor-homing phage may carry out combination by excretory CRKL.

In view of CRKL does not have hydrophobicity N-end sequence to carry out protein excretion people such as (, 1984) Walter via endoplasmic reticulum and the dependent Secretory Pathway of golgi body, whether stop the cell of CRKL to discharge so measured classical secretion inhibitor.The result of these researchs shows: inhibitor for example brefeldin A (Brefeldin A) and thapsigargin (thapsigargin) does not suppress the secretion of CRKL; In contrast, the inhibitor U26452 (glybenclamide) of discovery atp binding cassette (ABC) translocator stops the release of CRKL really.Have at least 4 kinds of known procedures to realize " secretion of the albumen of no classical signals peptide (comprising some somatomedin and cytokine) " (Nickel, 2003; People such as Prudovsky, 2003).These data declarations CRKL may be secreted via non-classical output pathway by abc transport albumen.It should be noted that for example interleukin-1 ' beta ' and macrophage migration inhibitory factor also use ABC-translocator system (people such as Marty, 2005 to other biological activity somatomedin; People such as Flieger, 2003; People such as Hamon, 1997), this has proposed the hypothesis of possible active transport mode.But result provided herein does not get rid of following possibility: necrocytosis-and no matter be a part as clonal selection in the malignant progression process, or after the cytotoxicity chemotherapy-also can in tumor microenvironment, produce extracellular CRKL.In order to obtain further understanding, designed in conjunction with measuring to describe CRKL, β in detail about this albumen composition ₁Biochemistry between integrin and the similar peptide of PSI structural domain interacts.By using two kinds of multi-form recombinant C RKL (being gst and his-label), presentation of results CRKL in fact can same dimerization, may be to carry out (people such as Kishan, 1997 by the SH3 structural domain; People such as Kristensen, 2006).Because intracellular CRKL participates in map kinase and 6 integrin-mediated approach (people such as Li, 2003; People such as Uemura, 1999), so detected phosphorylated protein in these 2 kinds of approach.When external when tumour cell adds recombinant C RKL, found the albumen of several phosphorylations, comprise paxillin, p130Cas, Erk1, Erk2 and Elk1, even also have CRKL itself.In addition, when to β ₁Integrin siRNA strikes when adding the recombinant C RKL of external source in the low cell, has found the Erk1 and the Erk2 albumen of the phosphorylation of minimizing, and this shows that external source rCRKL depends on β to the activation of ERK approach ₁The expression of integrin.Positive control mitogen is used to activate the dependent approach of map kinase and stimulates the DU145 tumour cell, strikes in the low cell so that be presented at CRKL siRNA, still keeps the overall efficacy of cell signaling mechanism.These results prove that excretory CRKL can activate 6 integrin-mediated and map kinase approach.

SiRNA research

CRKL (mRNA registration number NM_005207), β ₁Integrin (mRNA registration number NM_002211) and the contrast siRNA available from Santa C ruz, Ambion and Dharmacon.SiRNA oligonucleotide sequence and corresponding manufacturer are summarized in table 3.Use Oligofectamine (Invitrogen) or DharmaFect (Dharmacon) that the siRNA transfection is entered DU145 cell (1-2x10 ⁵Individual cells/well).Before handling with cells transfected incubation 48-72 hour.In cells transfected, do not observe necrocytosis or apoptosis, such as by Annexin-V dyeing (Roche) mensuration.Collect cells transfected and carry out cracking under the situation of proteinase inhibitor existing.Also strike the combination activity that has detected the tumor-homing phage in the low cell at CRKL.For saving experiment, the seedbed adds the recombinant C RKL that reaches 1.5 μ g outside CRKL siRNA cells transfected.The His-label recombinant C RKL albumen (400 η g/ml) of external source or reference protein (EGF, 200 η g/ml; MIF, 300 η g/ml; PMA, 300 η g/ml) be used for β ₁Integrin strikes low experiment.Load the albumen of equivalent and, use suitable antibody then by the Western engram analysis by the SDS-PAGE separation.Use WST-1 (Roche) to carry out cell proliferating determining.Use Boyden cell assay method (Corning) to carry out cell migration assay.

Table 3:siRNA

Santa?Cruz，CRKL：
	GUCGUAUUGUCAAAGAGUATT(SEQ?ID?NO：54)
GUAGCAGACAACACACAAATT(SEQ?ID?NO：55)
	CAGCAGACCUAGAAAUGUATT(SEQ?ID?NO：56)
Dharmacon，CRKL：
	CCGAAGACCUGCCCUUUAAUU(SEQ?ID?NO：57)
GAAGAUAACCUGGAAUAUGUU(SEQ?ID?NO：58)
	GUCACAAGGAUGAAUAUAAUU(SEQ?ID?NO：59)
AAUAGGAAUUCCAACAGUUUU(SEQ?ID?NO：60)
	Ambion，CRKL：
GGUAUCCAAGCCCACCAAUTT(SEQ?ID?NO：61)
	GGAUGAAUAUAAAUGGCCATT(SEQ?ID?NO：62)
Santa?Cruz，β ₁Integrin:
	GAGAUGAGGUUCAAUUUGATT(SEQ?ID?NO：63)
GAUGAGGUUCAAUUUGAAATT(SEQ?ID?NO：64)
	GUACAGAUCCGAAGUUUCATT(SEQ?ID?NO：65)
Dharmacon, contrast:
	AUGAACGUGAAUUGCUCAAUU(SEQ?ID?NO：66)

Immune precipitation determination

With cell in the RPMI serum free medium 24-48 hour synchronously, get off by centrifugation then, and the filter paper filtering by 0.22 μ m, preadsorption is to control antibodies.In the supernatant liquor that reclaims, add isopyknic PBS, use suitable antibody to carry out immunoprecipitation then.Brefeldin A, U26452 and thapsigargin are used for secretion and suppress research.In serum free medium with cell with compound incubation 9 hours, use suitable antibody to carry out immunoprecipitation then.In immunoprecipitation research, do not use stain remover.By using aforesaid method, discovery CRKL is in the solvable state of free rather than is in the vesica.

The cancer target that the part of embodiment 7CRKL mediation instructs

Next assessed in carrying the mouse of tumour cancer target characteristic in conjunction with the phage of CRKL.At first, designed and produced show selected in conjunction with the peptide of CRKL or the phage construct of one group of control peptide (mutant or messy).In human tumour heterogeneity graft (Kaposi sarcoma KS1767 cell and prostate cancer DU145 cell) and isogenic mouse tumor model (EF43-FGF4 mammary cancer), tested phage clone.After the phage of systemic administration, observe significant and specific tumor-homing in conjunction with CRKL; On the contrary, the contrast construct does not show the location (Fig. 9 A-D) to tumour.As shown in Figure 9, peptide sequence P-＞A (YRCTLNSAFFWEDMTHECHA of sudden change; SEQ ID NO:25), messy 1 (YRFCTSPFHEWHLENTDMCA; SEQ ID NO:26) and messy 2 (YRECTDSPHEFHLWNTMCAF; SEQ ID NO:27) do not demonstrate cancer target.Peptide (the YRCTLNSPAAAEDMTHECHA that the proline residue place comprises FFW-＞AAA sudden change is adjoined in use in SEQ ID NO:1; SEQ ID NO:28) or 2 other messy sequence (#3:YRCETDSPHEFHLWNTMCAF; SEQ ID NO:29 and #4:YRCETDSPHEFHLWNTFCAM; SEQ ID NO:30) the other research of carrying out does not demonstrate the cancer target activity of mutant yet.In addition, studies show that: incubation is in conjunction with the phage of CRKL and then when the mouse of carrying tumour is used in advance when using recombinant C RKL, and tumor-homing is suppressed (Figure 10).Also find: compare with phage that (peptide sequence is CDCRGDCFC (SEQ ID NO:31), and it is known as RGD-4C in conjunction with alpha v integrin; It is often used as the positive control of this type of experiment) (people such as Hajitou, 2006; People such as Arap, 1998; People such as Pasqualini, 2001; People such as Giordano, 2001; People such as Javadpour, 1996; People such as Ellerby, 1999; People such as Pasqualini, 1997), exceed at least 10 times in conjunction with the phage of CRKL.

At last, design and carried out comprising the preliminary preclinical test (Figure 11) of the mouse of carrying big or small tumour of mating of 4 formations.The following reagent of animals received: (i) the similar peptide of synthetic PSI structural domain; The (ii) similar peptide of synthetic PSI structural domain that is connected with short apoptotic plan peptide (people such as Arap, 2004; People such as Ellerby, 1999; People such as Kolonin, 2004; People such as Zurita, 2004), so that after receptor-mediated internalization, induce the apoptosis of target; (iii) independent synthetic is urged apoptotic plan peptide; Or (iv) independent medium.All peptides or intend peptide all with etc. volumetric molar concentration use.The result shows in these bodies, and under the experiment condition of being assessed, the similar peptide of PSI structural domain does not have detectable effect for tumor growth, and the short apoptosis plan peptide of target significantly suppresses tumor growth.

In-vivo tumour target and inhibition

According to describing (people such as Hajitou, 2006; People such as Arap, 1998; People such as Arap, 2004; People such as Kolonin, 2004; People such as Marchio, 2004) use phage to carry out target experiment in the body.The animal of using in the experiment is: the male nude mouse of carrier DU145 heterograft, or the female nude mice of carrier's Kaposi sarcoma KS1767 deutero-heterograft (subcutaneous) and carry the female mouse of immunocompetent Balb/c of EF43-FGF4 deutero-breast tumor (mammary fat pad orthotopic transplantation).In brief, will carry tumour (～8mm) mouse anesthesia and by tail vein in the intravenously mode to every injected in mice 5x10 ¹⁰T.U. wild-type YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1)-phage, or negative control: fd-tet phage (not having the son of insertion) and messy YRFCTSPFHEWHLENTDMCA (SEQ ID NO:26)-phage, YRECTDSPHEFHLWNTMCAF (SEQ ID NO:27)-phage, YRCETDSPHEFHLWNTMCAF (SEQ ID NO:29)-phage, YRCETDSPHEFHLWNTFCAM (SEQ ID NO:30)-phage, or mutant YRCTLNSAFFWEDMTHECHA (SEQ ID NO:25)-phage and YRCTLNSPAAAEDMTHECHA (SEQ ID NO:28)-phage.Each group phage clone is accepted in formation with 2 mouse of big or small tumour of mating.After 24 hours,, and, proofread and correct according to the weight of tissue by infectation of bacteria recovery phage from every mouse tumor resection.For each tumor model, experiment repeats twice.For the inhibition of cancer target, at first CRKL is gone back to the nest phage and reorganization gst-CRKL or contrast gst albumen were 37 ℃ of incubations 30 minutes, and intravenously is applied to the mouse of carrying tumor of prostate then.The fd phage is as negative control; RGD-4C (people such as Hajitou, 2006; People such as Arap, 1998) as positive control.In paraffin-embedded tumor tissue section, by TUNEL dyeing (Promega), detect minimum background apoptosis only arranged (less than total cell 1%).

Treatment in carrying the mouse of tumour

The mouse of carrying the DU145 tumour is carried out the size coupling and is divided into each formation (every group of n=4).Synthesize and short apoptosis motif _D(KLAKLAK) ₂The tumor-homing peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) that merges.Unconjugated peptide YRCTLNSPFFWEDMTHECHA (SEQ ID NO:1) or _D(KLAKLAK) ₂With comparing.Systemic administration synthetic peptide is according to describing (people such as Arap, 1998; People such as Arap, 2004) the mensuration gross tumor volume.

Use the peptide of target CRKL to carry out the MRI imaging

With the phage (target CRKL) of target tumor and phage (in contrast) formation gold/phage imidazoles hydrogel that does not have insertion.In these hydrogels, mix ferric oxide with 30% final volume (by volume calculating).This hydrogel prepared product is used for MRI research subsequently, wherein in 3 mouse (DU145) tumours of carrying tumor of prostate the only AuFe of injection equivalent, contain the hydrogel (contain to have or not and insert sub phage) of the non-target of AuFe and contain the hydrogel of the target CRKL of AuFe.Can clearly see and the quantitative negative contrast that forms by the iron core mediation by MRI.

According to the disclosure, need not undo experimentation and can prepare and realize disclosed herein and claimed all compositions and method.Though with the formal description of preferred implementation the compositions and methods of the invention, but it will be apparent for a person skilled in the art that and not break away from notion of the present invention, spirit and scope and the step or the sequence of steps of composition described herein and method and this method changed.More specifically, it is evident that related reagent can be replaced reagent described herein on some chemistry and the physiology, and still obtains same or analogous result.Conspicuous for those skilled in the art all these type of similar replacements and modification all are considered to drop on as within spirit of the present invention, scope and the notion that claim limited of enclosing.

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Claims

1. the peptide that comprises the isolating target tumor of CRKL binding motif, described motif is defined as:

A) length is 6-20 amino acid;

B) and β ₁The similarity degree of the corresponding optimum match sequence alignment of integrin (SEQ ID NO:47) is at least 25%; And

The length of wherein said target peptide is 100 amino acid or amino acid still less, and is bonded to the cell of expressing CRKL under physiological condition.

2. the peptide of claim 1, wherein said CRKL binding motif and β ₁The similarity degree of the best-fit sequence alignment of integrin (SEQID NO:47) is at least 40%.

3. the peptide of claim 1, wherein said CRKL binding motif and β ₁The similarity degree of the best-fit sequence alignment of integrin (SEQID NO:47) is at least 50%.

4. the peptide of claim 1, wherein said CRKL binding motif and β ₁The similarity degree of the best-fit sequence alignment of integrin (SEQID NO:47) is at least 60%.

5. the peptide of claim 1, wherein said peptide have and are different from and β ₁The sequence of the best-fit sequence alignment of integrin (SEQID NO:47).

6. the peptide of claim 1, wherein said CRKL binding motif has and β ₁The best-fit sequence alignment in integrin (SEQ ID NO:47) PSI structural domain zone.

7. the peptide of claim 1, wherein said CRKL binding motif has and β ₁The best-fit sequence alignment in integrin (SEQ ID NO:47) PSI structural domain zone, described PSI structural domain zone is selected from amino acid/11 0 to 29; 15 to 34; 18 to 37; 36 to 55; 39 to 58; 45 to 64; 94 to 113; 196 to 215; 198 to 213; 203 to 222; 244 to 263; 330 to 349; 377 to 396; 379 to 398; 380 to 399; 398 to 417; 400 to 419; 413 to 432; 447 to 466; 460 to 479; 460 to 479; 464 to 483; 469 to 488; 474 to 493; 475 to 494; 512 to 533; 519 to 538; 551 to 570; 574 to 593; 577 to 596; 579 to 598; 590 to 609; 596 to 615; 613 to 632; 615 to 634; 616 to 635; 644 to 663; 648 to 667; 663 to 682; 674 to 693; 682 to 701; 721 to 740; 727 to 746; With 779 to 798.

8. the peptide of claim 1, wherein said CRKL binding motif has the sequence that is selected from SEQ IDNO:1 to SEQ ID NO:46.

9. the isolating peptide of claim 1, it is further defined as cyclic peptide.

10. the isolating peptide of claim 1, wherein said peptide is connected to molecule.

11. the isolating peptide of claim 10, wherein said molecule is an albumen, and described peptide put together or merge to described albumen to form protein conjugate, wherein said protein conjugate is not naturally occurring albumen.

12. the isolating peptide of claim 11, wherein said peptide are positioned at described proteic end.

13. being short natural death of cerebral cells agent, anti-angiogenic agent, cytokine, cytotoxic reagent, medicine, chemotherapeutics, hormone, somatomedin, microbiotic, antibody or its fragment or strand, survival factors, anti-cell, the isolating peptide of claim 10, wherein said molecule transfer die agent, hormone antagonist, antigen, peptide, albumen, diagnostic reagent, radio isotope or preparation.

14. the isolating peptide of claim 13, wherein said molecule are to be selected from following short natural death of cerebral cells agent: linear gramicidins, magainin, mellitin, alexin, cecropin, (KLAKLAK) ₂(SEQ ID NO:48), (KLAKKLA) ₂(SEQ ID NO:49), (KAAKKAA) ₂(SEQ ID NO:50), (KLGKKLG) ₃(SEQ ID NO:51), Bcl-2, Bad, Bak, Bax and Bik.

15. the isolating peptide of claim 14, wherein said short natural death of cerebral cells agent is (KLAKLAK) ₂(SEQ ID NO:48).

16. the isolating peptide of claim 15, wherein said SEQ ID NO:48 is made up of D amino acid.

17. the isolating peptide of claim 13, wherein said molecule are to be selected from following anti-angiogenic agent: thrombospondin, angiostatin, the pigment epidermal derived factor, angiotensin, the ln peptide, the fibronectin peptide, inhibitors of plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, Interferon, rabbit, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, the 2-methoxyestradiol, the proliferin associated protein, carboxylic amine triazole, CM101, Marimastat, the many vitriol of piperylene, angiogenin 2, Antibiotic TAN 420F, PNU145156E, 16K prolactin antagonist fragment, linomide, thalidomide, pentoxifylline, genistein, TNP-470, Endostatin, taxol, Docetaxel, polyamine, proteasome inhibitor, kinase inhibitor, signaling peptides, accutin, cidofovir, vincristine(VCR), bleomycin, AGM-1470, platelet factor 4, MINOCYCLINE HCL, Endostatin XVIII, Endostatin XV, the terminal Hemopexin structural domain of the C-of matrix metalloproteinase-2, kringle 5 structural domains that human plasmin is former, the fusion rotein of Endostatin and angiostatin, the fusion rotein of kringle 5 structural domains that Endostatin and human plasmin are former, the monokine that interferon-induces (Mig), the fusion rotein of Mig and IP10, solubility FLT-1 (fins sample Tyrosylprotein kinase 1 acceptor), or kinases inserts minor structure domain receptor (KDR).

18. the isolating peptide of claim 13, wherein said molecule are to be selected from following cytokine: interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-(IF-γ), IF-α, IF-β, tumour necrosis factor or GM-CSF (rHuGM-CSF).

19. the isolating peptide of claim 10, wherein said peptide is connected to macromolecular complex.

20. the isolating peptide of claim 19, wherein said mixture are virus, phage, bacterium, liposome, microparticle, magnetic pearl, yeast cell or mammalian cell.

21. the isolating peptide of claim 13, wherein said peptide is connected to virus.

22. the isolating peptide of claim 14, wherein said virus are slow virus, papovavirus, adenovirus, retrovirus, AAV, vaccinia virus or simplexvirus.

23. the isolating peptide of claim 19, wherein said peptide is connected to solid carrier.

24. the isolating peptide of claim 23, wherein said solid carrier are microtitration ware or microchip.

25. prepare the method for construct, it comprises: obtain peptide according to claim 1; With described peptide is connected to molecule with the preparation construct.

26., said method comprising the steps of with peptide, molecule or proteic method of sending the cell of targeted expression CRKL:

(a) obtain according to claim 1 to 24 each peptide or the peptide of the method preparation by claim 23; With

(b) described peptide is applied to cell colony, wherein said colony comprises the cell of expressing CRKL, thereby molecule or albumen are delivered to described cell.

27. the method for claim 26, the cell of wherein said expression CRKL is in the experimenter, and peptide or albumen fusion constructs are formulated in the pharmaceutically acceptable composition, and described composition is applied to the experimenter.

28. the method for claim 27, wherein said experimenter is the human experimenter.

29. the method for claim 26, wherein said method is further defined as detection method, and described method comprises that also detection has been delivered to the peptide of cell, molecule or albumen.

30. the method for claim 26, wherein said experimenter has disease or illness, and described method is further defined as methods of treatment.

31. the method for claim 29 or 30, wherein said experimenter has cancer.

32. the method for claim 31, wherein said cancer are selected from prostate cancer, mammary cancer, sarcoma, gingival carcinoma, tongue cancer, lung cancer, skin carcinoma, liver cancer, kidney, cancer eye, the cancer of the brain, leukemia, mesothelioma, neuroblastoma, a cancer, neck cancer, carcinoma of the pancreas, kidney, osteocarcinoma, carcinoma of testis, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, the cancer of the brain, colorectal carcinoma and bladder cancer.

33. the method for claim 32, wherein said cancer is a prostate cancer.

34. the method for claim 32, wherein said cancer is a mammary cancer.

35. the method for claim 32, wherein said cancer is a sarcoma.

36. the process of claim 1 wherein that it is 6 to 10 amino acid that described motif is further defined as length.

37. the process of claim 1 wherein that it is 14 to 20 amino acid that described motif is further defined as length.